# Identification of conserved sequences within tospovirus genomes for broad-spectrum resistance using RNA interference (RNAi)

J. OLIVER (1), D. Rotenberg (1), A. Whitfield (1)

(1) Kansas State University, U.S.A.

Tomato spotted wilt virus (TSWV) and other tospoviruses cause significant yield losses in numerous crop species worldwide. Genetic resistance is the most effective management tool for tospovirus diseases in tomato; however, resistance breaking isolates of TSWV have emerged which are capable of overcoming the currently deployed resistance genes. An alternative to traditional resistance genes is transgenic resistance through the process of RNA interference (RNAi). Though effective against tospoviruses, the sequence-specific nature of RNAi means that resistance can break down if host plants are challenged with genetically diverse populations of tospoviruses. To examine viral sequence diversity, we used publicly available viral sequences of TSWV and other tospoviruses. Our approach focused on identifying short, highly conserved regions among 2679 TSWV sequences and across 589 additional sequences from seven tospovirus species. This analysis allowed us to locate sequence conservation within each of the five tospovirus genes. The identified sequences corresponded to motifs of known biological importance. Based upon their high conservation at the nucleotide level, correspondence with known biological functions, and low potential for off-target effects in tomato, these sequences have been incorporated into anti-viral RNAi constructs for tomato transformation.

# Correlation of endogenous pararetrovirus with symptoms of citrus blight disease

A. ROY (1), J. Hartung (2), A. Stone (1), J. Sharo (2), R. Brlansky (3), W. Schneider (1)

(1) USDA-ARS-FDWSRU, U.S.A.; (2) USDA-ARS-MPPL, U.S.A.; (3) University of Florida, CREC, U.S.A.

Citrus blight (CB) is a major economic problem in citrus groves in Florida. Even though the disease was first described in Florida in 1894 the causal agent of CB is still unknown. A number of biotic and abiotic agents have been postulated as potential causes of CB. CB occurs in a wide variety of locations with diverse environments and is root graft transmissible, suggesting that the disease is caused by a systemic infectious biotic agent. Recently, we documented a population of endogenous pararetrovirus (EPRV) sequences in a root stock susceptible to CB, carrizo citrange. Five EPRV sequences were obtained from healthy, huanglongbing (HLB)-infected and blight-affected carrizo citrange, but only one pararetrovirus sequence, termed Citrus blight associated pararetrovirus (CBaPRV), was found as RNA in all CB affected plants but not in any of the HLB diseased or healthy plants. To assess the correlation between active CBaPRV RNA and CB, samples from 8 healthy trees and 42 citrus trees diagnosed as blighted using the syringe injection technique were collected from four locations in Florida. Reverse transcription-PCR with primers specific to CBaPRV amplified the active RNA from CB affected roots, leaves, bark and flowers, but not from any healthy or HLB infected tissues. While there is no direct evidence that CBaPRV is the causal agent of blight, the presence of viral RNA is tightly correlated with CB symptoms.

# Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato

X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1)

(1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A.

Since its first discovery in 2013 in Mexico, Tomato mottle mosaic virus (ToMMV), a new tomato-infecting tobamovirus is now present in a number of countries (i.e., Brazil, China, and Israel) and several states in the U.S. There is little information available on the molecular and biological properties of this emerging virus. With approximately 80-85% nucleotide sequence identity to two other common tobamoviruses, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), there is a need to understand the genetic diversity and serological and biological properties of this emerging virus in order to develop a species-specific detection method, and to assess disease resistance durability in tomato cultivars. Comparative host range evaluation showed similarity in host species infected by three tobamoviruses. However, ToMMV was more aggressive on host plants than that of TMV and ToMV. To evaluate whether the emerging ToMMV could break the disease resistance, three commercial tomato cultivars were screened along with control Moneymaker tomato through mechanical inoculation. We observed different responses in tomato cultivars to the three tomato-infecting tobamoviruses. ToMMV exhibited aggressiveness and broke the resistance in a tomato cultivar that had demonstrated full resistance to both TMV and ToMV. This resistance breaking in tomato by the emerging ToMMV presents a serious concern for tomato growers. The mechanism of such resistance breaking will be discussed.

# Diversity of aflatoxin-producing fungi in non-agriculture soils of Zambia

P. KACHAPULULULA (1), J. Akello (2), R. Bandyopadhyay (3), P. Cotty (4)

(1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Lusaka, Zambia; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) USDA-ARS/University of Arizona, Tucson, AZ, U.S.A.

Aflatoxins are cancer-causing, immunosuppressive toxins that contaminate maize and groundnut, important staples in Zambia. Aspergillus flavus, A. parasiticus and two unnamed taxa are frequently implicated as causal agents of contamination in Africa. Most of Zambia's arable land is uncultivated. Aspergillus communities in crops may have originated in uncultivated regions and soils in these areas may serve as reservoirs of toxin-producers. Understanding fungal communities in cultivated and uncultivated areas may facilitate aflatoxin management. Soils from fields cropped to maize and groundnut (161) and from uncultivated land (53) were assayed for Aspergillus section Flavi from 2012 to 2016. Communities in both wild and cultivated soils were dominated by A. parasiticus (90% and 50%, respectively) and the A. flavus L morphotype (5% and 31%, respectively). Phylogenetic analyses with partial gene sequences for nitrate reductase (niaD, 1.8 kb) and the aflatoxin pathway transcription factor (aflR, 2.1 kb) resolved 20 A. parasiticus isolates from cultivated soils into 4 well supported lineages. Relationships among the 4 lineages and A. parasiticus from other regions were evaluated. Impacts of fungi present prior to introduction of crops may cause differences in the etiology of aflatoxin contamination among regions. Characterization of fungi from non-cultivated soils may provide insight on which fungi are best adapted to the region and allow for improved aflatoxin management.

# Differential effects of DON and 15-ADON on maize seedling growth

Y. MENG (1), J. Li (2), G. Munkvold (3), C. Liang (2), L. Luo (2)

(1) China Agricultural University; Iowa State University; Hexi University, China; (2) China Agricultural University, China; (3) Iowa State University, U.S.A.

Fusarium graminearum (Fg) is a common pathogen causing maize seedling blight in China. Trichothecene mycotoxins produced by Fg have been implicated as pathogenicity factors in seedling blight, but little is known about the effects of specific toxins, such as DON and 15-ADON. In order to study the effect of both mycotoxins on seed germination, maize seeds were soaked in 20 µg ml⁻¹ of DON or 15-ADON for 12h and germinated by rolled paper method; seed germination was counted daily and root length and seedling weight were measured after 7 days. The results showed both mycotoxins decreased seed germination significantly compared to the control, but there was no significant difference between DON and 15A-DON treatments. However, 15 A-DON displayed significantly reduced root length and lateral root numbers compared to DON. We speculated that 15-ADON production by F. graminearum in early stages of infection plays an important role in the poor development of roots, which is a typical symptom of maize seedling blight in the field.

# Diversity of aflatoxin producing fungi in Malawi

C. CHING'ANDA (1), J. Atehnkeng (2), R. Bandyopadhyay (3), P. Cotty (4)

(1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Lilongwe, Malawi; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) USDA-ARS, The University of Arizona, School of Plant Sciences, Tucson, U.S.A.
Aflatoxins are toxic fungal metabolites that often contaminate Malawi's food and feed. These toxins cause immune suppression, stunting, and cancer. *Aspergillus flavus* and *A. parasiticus* are the most frequently reported causes of contamination. Aflatoxin producers vary widely in genetic diversity, aflatoxin production, and agro-ecological preference. Little is known about the diversity of fungi causing aflatoxin contamination in Malawi. The current study examined DNA variability among aflatoxin producers in Malawi. Maize and groundnuts were sampled across the five agro-ecological zones of Malawi and fungi infecting crops and belonging to *Aspergillus* section *Flavi* were identified. The *A. flavus* L morphotype comprised 44% of the infecting fungi. Fungi with S morphology composed 40% and *A. parasiticus* made up 15%. Use of molecular phylogenetics to assign S morphology fungi to species is underway. Fungi that did not produce aflatoxins (793) and belonged to the *A. flavus* L morphotype were characterized at 17 microsatellite loci in order to identify fungi of potential value as biocontrol agents for the aflatoxin prevention. 350 microsatellite haplotypes were detected. Knowledge of variability among the aflatoxin producers may also provide opportunity to improve aflatoxin management.

## Effects of nutrients and microorganisms on aflatoxin production by *Aspergillus flavus* during successive generations of subculturing

O. OMOLEHIN (1), Y. Ruarang (2), R. Brown (3), Z. Chen (2)
(1) Louisiana State University, U.S.A.; (2) Department of Plant Pathology and Crop Physiology, Louisiana State University, U.S.A.; (3) Southern Regional Research Center, USDA-ARS., U.S.A.

*Aspergillus flavus* infects major agricultural crops, and produces aflatoxin which has been linked to increased liver cancer in humans. Factors such as nutrition, pH and temperature are known to influence *A. flavus* growth and subsequent aflatoxin production. However, the long-term effects of various media and the presence of other microorganisms on aflatoxin production have not been well studied. In the present study, *A. flavus* AF13 strain was grown on media containing different levels of nutrients, such as V8, Potato Dextrose Agar (PDA) and Luria Broth (LB), and subcultured for 56 generations. The highest aflatoxin production was observed with *A. flavus* grown on PDA, which was about 100 times higher than on LB or V8. Aflatoxin production appears to increase with successive transfers of *A. flavus* on the PDA medium. On the other hand, aflatoxin production seems to decrease slowly with subsequent transfer when *A. flavus* is grown on V8 or LB medium. At the 39th generation on LB medium, aflatoxin production decreased to a level that was undetectable with HPLC. *A. flavus* that produced no detectable aflatoxins on LB medium regained the ability to produce high levels of aflatoxin when transferred from LB to PDA medium. These results confirmed earlier studies which have demonstrated that nutrients, especially the carbohydrates in PDA, play a key role in aflatoxin production over time. The impact of microorganisms on aflatoxin production in LB and V8 media will be discussed.

## Mycotoxins contamination on hybrids and landraces of stored maize grain in México

L. VASQUEZ-SILLER (1), K. Ordóñez-Morales (2), J. Soria-Ruiz (3)
(1) Universidad Autónoma Agraria Antonio Narro, Mexico; (2) Semillas Berestain, Mexico; (3) Instituto nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP), Mexico

Mycotoxins contamination is a human and animal health problem because the exposure to such substances present on grains infected by several fungi can cause diseases; thus this work explored mycotoxins contamination levels on maize grain in México. A survey was conducted in six locations forming three sets of samples including hybrids and landraces collected by UAAAN, a seed enterprise and cooperating farmers. 31 samples were tested for moisture content (MC) by constant temperature oven method; phytopathogenic fungi detected and counted by freeze blotter test, and aflatoxins B1, total fumonisins and deoxynivalenol contamination levels determined with inverted ELISA test (Enzyme-Linked-Immune-Sorbent-Assay) (Romer Labs®). General mean of MC was 11.63%. Potentially toxigenic phytopathogenic fungi detected were Fusarium verticillioides 7.6%, Fusarium graminearum 2%, Aspergillus spp. 9.2% and Penicillium spp. 1.1%. Mycotoxins levels were 0.106-2.665 ppm of total fumonisins, 0.64-2.67 ppm of deoxynivalenol and 0 ppb of aflatoxins B1. Mycotoxins levels detected on maize grain were safe for consumption according to Food and Drug Administration (FDA) recommendations, which are: 2-4 ppm of total fumonisins, 5-10 ppm of deoxynivalenol and 20 ppb of aflatoxins B1. Some mycotoxins detection levels were near the FDA risk values, indicating the need to regulate also other mycotoxins than total aflatoxins, which currently is the only one regulated by the Government of México.

## Profile of Aspergilli in fields under continuous maize and groundnut cultivation and aflatoxin contamination along the crop value chains in Mozambique

J. AUGUSTO (1), J. Atehnkeng (2), J. Akello (3), P. Cotty (4), R. Bandyopadhyay (5)
(1) Intl Inst of Tropical Agriculture, Nampula, Mozambique; (2) International Institute of Tropical Agriculture, Lilongwe, Malawi; (3) International Institute of Tropical Agriculture, Lusaka, Zambia; (4) University of Arizona, Tucson, AZ, U.S.A.; (5) International Institute of Tropical Agriculture, Ibadan, Nigeria

Aflatoxins are routinely reported in maize and groundnut in Mozambique, but the profile of aflatoxigenic fungal isolates in the fields continuously cultivated with maize or groundnut and the extent of aflatoxin accumulation along the maize and groundnut value chains are unknown. Georeferenced soil samples were taken from 250 farmers' fields at harvest in 2013 and 2014 in different agro-ecological zones (AEZ) to determine Aspergilli profile. The fields had been under continuous maize or groundnut cultivation from two to five years. Also, aflatoxin content was measured in 300 maize and groundnut samples collected along the crops' value chains. Inoculum density of Aspergilli was high (>2,000 CFU/g) in fields under extended (³3 years) continuous cultivation, and low-medium (0-2,000 CFU/g) either in fields with <3 years of consecutive cultivation, or at high altitude AEZ (>1,000m), irrespective of crop. *Aspergillus flavus* S (34%) and L (31%) morphotypes were most frequently isolated from fields under intensive cultivation (³3 years), but L morphotype was predominant (>50%) in fields with <3 years of continuous cultivation, irrespective of crop or AEZ. Aflatoxin content was high in groundnut leftovers used for human consumption after sorting (max. 3,874 ppb), groundnut from storage (max. 1,777 ppb) and groundnut sold at marketplaces (max. 720 ppb). These results suggest that targeted aflatoxin management practices may be required along the crop value chains in Mozambique.

## Influence of corn GMO traits for insect control on aflatoxin and fumonisin contamination in Texas

T. ISAKEIT (1), T. Isakeit (1), S. Murray (2), J. Pekar (2)
(1) Texas A&M University, U.S.A.; (2) Texas A&M University, U.S.A.

Contamination of corn with aflatoxin and fumonisin is a chronic problem throughout most of Texas. Insect damage to ears can be a predisposition factor, so controlling insects using GMO traits could be a management tool. The objective of our field experiments was to evaluate the impact of three different GMO traits in one parent hybrid (N78S) on mycotoxin contamination. Hybrids were planted in two-row, 22 ft-long plots in a randomized, complete block design near College Station in 2014 and 2015 and were inoculated with either *Aspergillus flavus* or *Fusarium verticillioides* or both. Overall, in 2014, the Viptera-trait hybrid expressing *Cry1Ab* and *Vip3Aa20* Bt proteins accumulated less aflatoxin (28 parts per billion (ppb) average, range 1-84 ppb) than hybrids that expressed only *Cry1Ab* (161 ppb, range 38-340 ppb), or no Bt proteins (175 ppb, range 54-410 ppb). Fumonisin was significantly (P=0.05) reduced in the Viptera-trait hybrid (0.3 parts per million, ppm) compared with the other hybrids (5.5 ppm and 7.5 ppm). In 2015, there was greater drought stress at flowering, which increased overall aflatoxin levels. The Viptera hybrid had the lowest average aflatoxin, 147 ppb, compared with the other hybrids (346 ppb and 274 ppb). The average fumonisin was 3 ppm with the Viptera hybrid, compared with the other hybrids (9.5 ppm and 12 ppm). These results show that in some years, the insect-control traits in some GMO hybrids will not be sufficient to reduce aflatoxin.
## Conidial versus sclerotial production: Implications for corn and soil niche specialization in *Aspergillus flavus*

R. SWEANY (1), C. DeRobertis (1), K. Damann (1)

(1) Louisiana State University Agricultural Center, U.S.A.

*Aspergillus flavus* contaminates corn and other oil-seed crops with carcinogenic aflatoxin. The populations of *A. flavus* differ between soils and crops. In Louisiana, native *A. flavus* isolated from soil and corn in 11 fields revealed only 6 of 16 vegetative compatibility groups (VCG) infected corn. During inoculum production for an infectivity titration experiment to verify niche specialization, two predominant VCGs in corn produced approximately 15 times more conidia than the most widespread soil VCG. Instead of producing conidia, the widespread soil VCG produced copious sclerotia. To test the hypotheses that corn infecting strains produce more conidia and soil inhabiting strains more sclerotia, three isolates from each of 16 VCGs were grown on Czapek Dox and Wickerham solid media and assessed for conidial or sclerotial production respectively. Generally corn VCGs produced more conidia; two VCGs comprising 91% of corn isolates produced significantly more conidia (3-60X) than 9 of 10 strictly soil VCGs. Conversely, those two corn VCGs produced very limited sclerotia, whereas all strictly soil VCGs produced significantly more sclerotia (10-100X). High conidial production appears to correlate with corn infection, whereas production of resting bodies appears to correlate with survival in the soil. Suppression of conidial production toward sclerotial production may reduce corn infection and limit outbreaks of aflatoxin contamination in corn.

## Evaluation of sorghum germplasm for resistance to grain mold and mycotoxin contamination in the Mid-Atlantic

B. ACHARYA (1), N. McMaster (2), M. Balota (1), D. Schmale (2), H. Mehl (1)

(1) Virginia Tech Tidewater AREC, U.S.A.; (2) Virginia Tech, U.S.A.

Grain sorghum production is increasing in the Mid-Atlantic region due to demand for locally sourced animal feed. Grain mold, caused by a complex of fungal species, infects grains, deteriorates quality, and reduces yield 30-100%. Some grain mold fungi produce mycotoxins that are toxic to both humans and animals, but little is known about the fungi and mycotoxins associated with sorghum grain in the Mid-Atlantic. The objective of this study was to evaluate sorghum germplasm for susceptibility to grain mold and mycotoxin contamination. Under natural field conditions, 375 genetically diverse sorghum accessions from more than 25 countries were evaluated for agronomic traits (panicle type and grain color), grain mold severity, and deoxynivalenol (DON) contamination. Mold severity ranged from 0-90%, and DON ranged from undetectable to 10 ppm. Grain mold severity and DON concentrations varied among panicle types and grain colors (P<0.001). Overall, open panicles with dark colored grains had lower mold severity and DON concentrations than compact panicles with light colored grains. Given the potential for grain mold and DON contamination in sorghum grown in the Mid-Atlantic, future work will focus on characterization of the causal agents of grain mold and their associated mycotoxins and identification of sorghum germplasm and agronomic traits associated with reduced susceptibility to mold and mycotoxin contamination.

## Seasonal population dynamics of mycotoxigenic *Aspergillus* section *Nigri* species on grapes and in vineyard soil using ddPCR

J. PALUMBO (1), T. O'Keeffe (1), M. Fidelibus (2)

(1) USDA ARS, U.S.A.; (2) University of California, U.S.A.

Several species of *Aspergillus* section *Nigri* (black Aspergilli), including mycotoxin producers, are common residents of grape vineyards, but measuring relative population sizes of individual species using traditional isolation and identification methods is impractical. We developed species-specific primers and probes for quantitative droplet digital PCR (ddPCR) to simultaneously detect and quantify *A. niger*, *A. carbonarius*, *A. welwitschiae* and *A. tubingensis* populations from grape and soil samples. Samples were collected at berry set, veraison, harvest and raisin stages from two vineyards over two consecutive years. Plate counts showed that total fungal counts were $10^2-10^3$ cfu/g on berries and $10^4-10^5$ cfu/g in soil. Plate counts of black Aspergilli in soil were between $10^2$ and $10^4$ cfu/g, independent of sampling time, while counts of black Aspergilli on berries increased from <$10^1$ cfu/g at berry set to >$10^3$ cfu/g on raisins. Distribution of *Aspergillus* species using ddPCR showed that in soil, relative abundance of each species varied widely among samples, but *A. niger* was the most abundant species in 52 of 60 samples. On grapes, relative abundance of *A. carbonarius* was higher on mature grapes and raisins, and correlated with ochratoxin levels in those samples. This ddPCR method is a useful tool to describe fungal population dynamics in the environment and to monitor mycotoxigenic *Aspergillus* species during grape and raisin production.

## Factors influencing *Aspergillus flavus* community structures in fields treated with the atoxigenic biocontrol *A. flavus* AF36

R. JAIME (1), P. Cotty (2)

(1) University of Arizona, U.S.A.; (2) USDA-ARS, University of Arizona, U.S.A.

Aflatoxins, toxic and carcinogenic metabolites produced by several fungi in *Aspergillus* section *Flavi*, are perennial contaminants of cottonseed in Arizona and impact crop value. Atoxigenic strains of *A. flavus* have been used to displace aflatoxin producers and manage aflatoxins in cottonseed in Arizona for over two decades. Previous work indicated applications benefit both treated and subsequent crops and that atoxigenic strains in treated soils decrease over time to reach equilibrium with the native *A. flavus* community. Crop rotation has been implicated as a factor influencing atoxigenic strain retention. The present study sought to determine factors influencing both retention of the biocontrol AF36 and reestablishment of the S strain. Weather, soil type and geography were all found to influence the community structure of *A. flavus* with hot temperatures, heavy soils, and low elevations favoring the S strain. Regression analyses indicated significant differences among regions in rate of change of the populations of both AF36 and the S strain as a function of time after application. Populations of *A. flavus* in soils are highest immediately after harvest. Steep declines occur during the few months following harvest. Both the S strain and AF36 followed similar patterns, fitting an exponential decay model. However, quantities of S strain propagules decline slower than AF36, suggesting the S strain may be adapted to resist soil factors that reduce *A. flavus* viability.

## Correlation between the disease incidence of Fusarium head blight and the mycotoxin content of winter cereal crops in Korea

H. SHIM (1), H. Shim (2), I. Kang (2), D. Shin (2), J. Roh (2), S. Heu (2)

(1) National Institute of Crop Science, South Korea; (2) National Institute of Crop Science, South Korea

In 2015, diseased grain rates of Fusarium Head Blight of winter cereal crops in southern region of Korea were 9.5% at an average and the incidence is slightly higher than compared to the previous year's. Diseased grain rates of Fusarium Head Blight on barley were 18.8% in Jeollanamdo province and that was the highest in Boseonggun with 26.4%. Diseased grain rates of Fusarium Head Blight on wheat was 19.2% in Jeollanamdo province and 12.9% in Jeollabukdo province. It showed the higher incidence in Haenamgun, Goseonggun especially with 35%. Mycotoxin content was investigated from the seed samples which have been collected by each region. Deoxynivalenol was 13.3 mg/kg in Sacheon city and 16.3 mg/kg in Goseonggun which was the higher disease incidence than the other regions. Zearalenone contents of the seed samples which have been collected in Sacheon city, Buangun, and Gangjingun as the highest incidence region were 1.15 mg/kg, 1.11 mg/kg, 1.62 mg/kg respectively. Fumonisin contents of the seed samples which have been collected in Sacheon city, Boseonggun, and Haenamgun were 2.54 mg/kg, 2.19 mg/kg, 2.64 mg/kg respectively. Correlation coefficient between the diseased grain rate of Fusarium Head Blight and the Zearalenone content was 0.81.

## Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with *Fusarium graminearum*

D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2)

(1) University of Manitoba, Winnipeg, MB, Canada; (2) North Dakota State University, Fargo, ND, U.S.A.

Mycotoxins are a chemically diverse group of toxic secondary metabolites produced by fungi. In order to cope with these mycotoxins, plants have the potential to modify the chemical structures as part of their defense mechanism. The resulting metabolic products are neither routinely screened at grain
elevators nor regulated by proper legislations. The objective of this study is to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content in Canadian spring wheat varieties grown in two locations, inoculated with different chemotypes of *Fusarium graminearum*. According to the analysis of variance, significant differences were observed among the varieties for fusarium head blight (FHB) disease index, fusarium damaged kernel percentage (%FDK), DON and D3G content. When considered the effect of chemotype, significant differences were observed for FHB disease index, %FDK and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes. Correlation analysis showed a strong positive correlation between DON and D3G. The highest D3G/DON ratio was observed in moderately resistant varieties. The susceptible varieties showed lower D3G/DON ratio compared to the moderately resistant varieties. The current study indicated that Canadian spring varieties produce D3G upon *Fusarium* infection and testing for D3G in food and feed safety assessments in Canada should be considered.

## Seed Quality of Maize Cultivars Infected With *Pantoea agglomerans*

H. SILVA-ROJAS (1), G. Morales-Valenzuela (1), L. Cordova-Tellez (1), D. Ochoa Martínez (1), A. Carballo-Carballo (1)

(1) Colegio de Postgraduados, Mexico

*Pantoea agglomerans* is a important seed borne pathogen of maize. There are reports about transmission rates, however, information concerning the quality seed is weak. The aim of this study was to evaluate the quality of seed produced in infected plants with *Pantoea agglomerans*. Dent corn hybrids Triunfo (comercial) and 9Bx52 (experimental) were sown in Experimental Station of Colegio de Postgraduados, Montecillo, Mexico. Disease assessments were made once a week during crops cycle. Seeds were harvested and separated according severity of mother plant (healthy, non-systemic and systemic). Seed quality was evaluated with standard test germination in laboratory and vigour in greenhouse. Disease incidences of systemic infection were 3.6% for Triunfo and 2.8% for 9Bx52. The results show that 1000-seed weight was lower in systemically infected plants. All variables in laboratory and greenhouse were significantly lower in seed plants with systemic infection. These results indicate that the severity of infection from the mother plant affects the quality of the seed.

## Rapid detection of preexisting internal *Leuconostoc* spp. spoilage populations in fresh-cut carrots during storage

K. BRITT (1), T. Suslow (1)

(1) UC Davis, U.S.A.

During an extended period of abnormally short quality retention in mixed component packaged salads, due primarily to rapid decay, a root-cause investigation was undertaken. From an initial investigative assessment, this study focused on identification of the primary underlying microbiological cause and suggested the need for a rapid detection screening of raw material. Analysis of several lots of unprocessed product, approx. 6 cm abrasively peeled carrot plugs, revealed the accumulation of an aqueous slime in the shipping bag void space and around the extremely softened plug surfaces. This premature diagnostic sign of lactic acid bacteria (LAB) spoilage, specifically Leuconostoc spp., developed in cold storage (2.5°C) after two weeks. Efforts were undertaken to determine whether the Leuconostoc was internalized in raw material or primarily environmental contamination with a proliferating reservoir of LAB in the primary processing and packaging environment. Polymerase chain reaction (PCR) primers specific for the amplification of a sequenced region of the Leuconostoc 16S ribosomal RNA gene confirmed the taxonomic identity. Total initial LAB and Leuconostoc bacterial populations isolated from symptomatic carrots ranged from log?? 7.5-8.5 and log?? 3.5-4.0 CFU/g carrot tissue weight respectively, and increased log?? 2 CFU/g log?? and 3.5 CFU/g respectively in population density on asymptomatic and symptomatic raw carrot material during two week refrigerated storage.

## Controlling bacterial soft rot in tomato fruit that have been inoculated through lenticels located around the stem attachment

J. BARTZ (1), D. Spiceland (1), M. Elkahky (3), J. Brecht (4)

(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Faculty of Agriculture, Mansoura University, Egypt; (4) Horticultural Sci.;

University of Florida, U.S.A.

Tomato fruit harvested from wet plants or exposed to rainfall prior to packing have a high potential for postharvest decays. In laboratory tests, bacterial soft rot (*Pectobacterium carotovorum*) development during storage of fruit inoculated in the stem depression was delayed by air-drying, wiping dry, or chlorine washes with efficacy linked with the interval between inoculation and treatment as well as type of treatment. Fruit that were exposed to water alone did not develop decay during storage. Incidence of bacterial soft rot was rare (0 to 13%) among inoculated fruit that were washed with chlorinated water, or wiped dry within 10 sec after dip inoculation, whereas lesions developed among > 50% of fruit washed or wiped dry at 60 min post inoculation. Incidence among non-treated controls was 100% within the 5-day storage. In three separate experiments, bacterial soft rot was reduced by an average of 52, 60, and 20% when fruit were air-dried (30 min) at 1, 4 or 18 h after inoculation, respectively. Corresponding values for washing in chlorinated water (200 ppm, pH 6.5) for 1 min averaged 48, 40 and 2%, respectively. Although evidence does not indicate that the pathogen is introduced into stem scar infection courts by contact such as occurs with wound inoculation, interventions must be initiated within an hour or less after stem scar exposure to inoculum in order to minimize the decay potential.

## Assessment of hermetic storage of maize under different environmental conditions

B. LANE (1), C. Woloshuk (1)

(1) Purdue University, U.S.A.

Storage of maize is a significant challenge for farmers in developing countries. Fungal growth and mycotoxin accumulation are some of the leading causes of postharvest losses, which are estimated to be $4 billion annually in the Sub-Sahara. One of the largest postharvest threats is the growth of *Aspergillus flavus* and the accumulation of aflatoxin. As maize production in Africa continues to grow, so does the need for storage methods that will help mitigate postharvest losses. This study investigated the use of Purdue Improved Crop Storage (PICS) bags, a three-layer hermetic-storage system, for the prevention of grain rewetting in humid environments in order to mitigate fungal growth and aflatoxin accumulation. Maize (14% grain moisture) was stored in PICS and woven polypropylene bags in Indiana and Arkansas, two environments contrasting in temperature and relative humidity. After 3 months of summer storage, moisture in the woven bags increased significantly in both environments (as high as 15.9%). Populations of storage insects and fungi also increased in the woven bags, with the greatest change under the Arkansas environment. In contrast, grain in the PICS bags was not affected by the two environments; grain moisture remained unchanged, there were no storage insects, and fungal populations increased only slightly. These results provide evidence that PICS bags can be used for the safe storage of maize in the highly-variable environment of Sub-Saharan Africa.

## Bitter rot on apple: Comparative epidemiology of *Colletotrichum* species and growth temperature

R. MOREIRA (1), E. Zielinski (2), A. Filho (3), L. May De Mio (2)

(1) Federal University of Parana, Brazil; (2) Federal University of Parana, Brazil; (3) University of Sao Paulo, Brazil

The *Colletotrichum* genus was reclassified, and based on this new classification were described five species causing Bitter Rot on apple belonging to two complex, *C. acutatum* complex (CaC: *C. nymphae*, *C. paranaense*, *C. melonis*) and *C. gloeosporioides* complex (CgC: *C. fructicola*, *C. siamense*). However, are unknown the optimum conditions for the development and the impact of each species in the disease progress. Therefore, the aim of this study was to determine, for each species, the optimum temperature and evaluate the infection and colonization in fruits of the cultivar Gala. The optimum temperature for the development of the colonies was 22-24° C for CaC, and 25-26°C for CgC. The progress of the disease was significantly higher in fruit inoculated with CaC than in fruit inoculated with CgC. The incubation period was 2 to 3 days for all isolates in wounded fruit, while in unwounded
the period was 18-25 days. The latency period was 7 days to *C. paranaense* and 6 days to *C. nymphae* with fruit wounded but in unwounded fruit, only *C. paranaense* spolulates with higher latency period (33 days).

## Use of induced resistance for the management of postharvest decay of fruit

G. ROMANAZZI (1)

(1) Marche Polytechnic University, Italy

Up to 25-30% of fruit harvested nowadays is lost, and decay causing fungi are among the main causes of such losses. Harvested produce has high economic impact, covering the costs of production, harvesting, transport, eventual cold storage, exposure on the shelf of stores (so called shelf life) and final life at the home of consumer. During this period, the fruit can be exposed to a list of fungi able to take profit of storage conditions, with high humidity, low temperatures, and senescence, that make weaker the fruit reaction to the infection. The main postharvest pathogen is *Botrytis cinerea*, but considerable losses of fruit are caused, according to the species, by *Penicillium*, *Colletotrichum* spp., *Moniliniae*, *Rhizpous* spp., *Mucor* spp., *Alternaria* spp., *Cladosporium* spp. and some other decay causing fungi. Nowadays the most frequent way to prevent postharvest decay relies in the use of synthetic fungicides, but considering their limitations, alternatives based on the use of biocontrol agents, physical means, decontaminating agents, and natural compounds are deeply studied and often used in the field and/or in packinghouses. Most of these alternative treatments have direct antimicrobial activity and at the same time they elicit the host defenses. The different ways to apply the induced resistance, with negative and positive aspects, mechanisms involved and tools useful to track the physiological changes occurring in the host tissues will be discussed.

## Susceptibility of sweetpotato (*Ipomoea batatas*) cultivars to fungal and bacterial diseases

E. PALENCIA (1), L. Quesada-Ocampo (1)

(1) North Carolina State University, U.S.A.

Sweetpotato [*Ipomoea batatas*], is the number one vegetable crop in the state of North Carolina. This crop is a high-nutrient content food and a great source of pro-vitamin A, vitamin C, calcium, and iron. Field and post-harvest diseases cause yield and economical losses during sweetpotato production. Rhizopus soft rot, caused by the ubiquitous *R. stolonifer*, Fusarium root rot, associated with *F. solani* and *F. oxysporum* f. sp. *batatas* infections, and Streptomyces soil rot, caused by *Streptomyces ipomoeae*, are among the most detrimental diseases of sweetpotato in North Carolina. Management of sweetpotato diseases through application of antimicrobial chemical compounds is limited since few products are labeled for use in sweetpotato and due to residue restrictions for export markets. Development of cultivars with genetic resistance is the most desirable and economical means of managing plant diseases. The present work was conducted to evaluate sweetpotato genotypes for resistance to economically relevant post-harvest diseases in sweetpotato production. For this purpose, replicated greenhouse and growth chamber experiments were performed to screen for resistance and susceptibility of 16 sweetpotato varieties to 8 microbial isolates. The results suggested that sweetpotato genotypes differed in resistance to the tested phytopathogenic isolates.

## Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit

L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3)

(1) Institut Valencià d'Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain

GRAS (generally regarded as safe) salts were preliminary selected in *in vitro* tests against *Botrytis cinerea*, the causal agent of tomato postharvest gray mold, and added at 2% wet basis (wb) to emulsion matrixes prepared with hydroxypropyl methylcellulose (HPMC) and glycerol (ratio 3:1 dry basis, db), beeswax (BW) and oleic acid (ratio 5:1 db), and tween 80 (1.5% wb). The final emulsion solid concentration was modified to 7-10% wb in order to obtain formulations with a viscosity range of 100-150 cp. Selected stable coatings were tested *in vivo* against gray mold on cherry tomatoes cv. Josefina artificially inoculated with *B. cinerea* 24 h before coating (curative activity) and incubated at 20°C for up to 15 days. Among these edible coatings, the most effective were those containing sodium propionate (SP), potassium carbonate (PC), ammonium phosphate (APh), and ammonium carbonate (AC), and they were further tested *in vivo* with fruit subjected to cold storage. Gray mold incidence and severity were determined on cherry tomatoes inoculated with *B. cinerea*, coated 24 h later, and stored at 5°C for 7 and 14 days followed by a shelf life period of 7 days at 20°C. The antifungal activity of the coatings was fungistatic rather than fungicidal. Although the HPMC-BW coatings formulated with SP were the most effective in reducing gray mold on cold-stored cherry tomatoes, coatings containing AC were selected due to their better performance for overall fruit quality preservation.

## Analysis of IAA and ABA in HLB diseased citrus trees

Z. PANG (1), H. Wang (1), N. Wang (1)

(1) Citrus Research and Educational Center, U.S.A.

HLB, caused by *Candidatus* Liberibacter asiaticus (Las), is a disastrous disease resulting in defoliation, twig dieback, root decline and fruit drop. Among these symptoms, preharvest fruit drop directly poses a disastrous threat to the citrus industry by decreasing yield. At early developmental stage of fruit, dropping always occur at AZ-A zone which is between branch and peduncle. Here, different hormones level at peduncle area were dynamically monitored both in healthy and HLB infected trees during the fruit maturation to find if hormone imbalance in HLB infected peduncles caused preharvest fruit drop. Indole-3-acetic acid (IAA) level was lower in HLB infected trees at the early stage of fruit ripening compared to healthy trees. Level of abscisic acid (ABA), which promote fruit mature and abscission, was higher in Las infected trees. Expression analysis of hormone related genes also matched the hormone detection. We will also report our results in preventing preharvest fruit drop using different hormones.

## Seasonal variation in the antibacterial activity of latex-like resin from *Sciadopitys verticillata*

K. GWINN (1), D. Yates (1), B. Ownley (1), N. Labbe' (1)

(1) University of Tennessee, U.S.A.

Japanese umbrella pine, *Sciadopitys verticillata*, produces a latex-like resin with antibacterial activity. To determine seasonal antibacterial activity, resin was collected in summer and winter of 2015, stored at -20°C, autoclaved twice, and suspended in sterile deionized water. Resin was tested within two months of collection. Bacterial suspensions (100 µl) from nutrient broth (NB) overnight cultures were mixed with NB (100 µl), treated with resin suspension, and incubated overnight at 26°C with shaking. Treatments were: 0 (control), 25, 50, and 100 µl resin suspension diluted in water (total volume 100 µl). Colony-forming units were determined with dilution plating. Separate tests were conducted with winter and summer-collected resins, and bacterial species were evaluated individually. Data (number of treatment colonies relative to control colonies) were analyzed with mixed models ANOVA. Differences were significant at *P*=0.05. Exposure to resin collected in winter reduced numbers of colonies of *Bacillus cereus*, *Erwinia amylovora*, *Agrobacterium tumefaciens*, and *Escherichia coli*, and increased numbers of *Xanthomonas campestris*, *Pseudomonas fluorescens* (Pf), and *Pseudomonas syringae*. Exposure to resin collected in summer affected population growth of two bacterial species; colony counts of *E. amylovora* decreased and those of *P. fluorescens* increased. Future research will address the role of chemical composition and resin concentration on antibacterial activity.

## Bioactive effect of triterpene extracts in mix with *Pseudomonas* to control *Gaeumannomyces graminis* var. *tritici* on wheat plants

E. MOYA ELIZONDO (1), J. González (2), T. Quezada (2), G. Silva (2)

(1) Departamento de Producción Vegetal, Universidad de Concepción, Chile; (2) Universidad de Concepción, Chile
Extracts rich in saponins from quillay (Quillaja saponaria Mol.), an endemic Chilean native tree, and rhizobacteria of the genus *Pseudomonas*, which produce antimicrobial compounds such as 2,4-Diacetylphloroglucinol (2,4-DAPG), have been associated with reduction of the fungus *Gaeumannomyces graminis* var. *tritici* (Ggt), causal agent of take-all disease of wheat. However, reports about bioactivity between these bacteria and this natural triterpenoids are not available. Through *in vitro* and *in vivo* tests the interaction between both new sources of take-all disease control were assessed. Results showed that quillay extracts Vet SAP® (>90% of saponins) and QL1000® (~8% of saponins) controlled Ggt *in vitro* and had a differential effect according to their purity on populations of 2,4-DAPG-producing *P. protegens*, with Vet SAP® not affecting the bacterial growth at 800 ppm of quillay saponins. On wheat seedlings, Vet SAP® did not affect the bacterial populations of the three bacterial strains assessed, although, quillay saponins at concentration of 7,360 ppm reduced bacterial biofilm formation on the rhizosphere. Strains of *P. protegens* had a variable antagonistic activity on wheat seedling roots and kept their disease control ability when were combined with different concentrations of Vet SAP®. These results suggest that mixtures of the antagonistic bacteria and plant extract to control *G. graminis* var. *tritici* could be useful in developing a biopesticide.

### Protection of wheat against Zymoseptoria tritici using sugar beet extract-based resistance inducers

A. SIAH (1), S. Mejri (2), A. Ghinet (3), B. Rigo (3), C. Abuhaie (3), M. Magnin-Robert (4), B. Randoux (4), P. Reignault (4), P. Halama (2)
(1) Institut Charles Viollette (EA 7394), Institut Supérieur d'Agriculture, Lille, France; (2) Institut Charles Viollette (EA 7394), Institut Supérieur d'Agriculture, France; (3) Laboratoire de Pharmacochimie (INSERM U995-LIRIC unit), Hautes études d'ingénieur, France; (4) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d'Opale, France

In the current context of sustainable agriculture, the use of resistance inducers (RIs) is a promising approach to control crop diseases. A total of 73 putative RIs, including sugar beet (SB) extract-based products, phytohormones, vitamins, and amino acid-based RIs, such as GABA and BABA, were assessed for their efficacy on wheat against *Zymoseptoria tritici*, a major pathogen on this crop. The experiments were performed in the greenhouse using third-leaf stage plants of the susceptible cv. Alixan treated with each putative RI using foliar application two days before inoculation with the pathogenic strain T02596. Disease symptom scoring three weeks post-inoculation revealed significant reductions of leaf chlorosis and necrosis as well as sporulation (pycnidial density) in plants treated with saccharine, salicylic acid, abscisic acid, GABA, BABA, vitamins C and BX, probenazole and 33 SB extract-based RIs, such as RI2474, which was the most efficient molecule (up to 71% disease reduction). Among the 73 tested molecules, 25 displayed an *in vitro* inhibiting effect on *Z. tritici* growth. The three most efficient SB extract-based RIs without biocide effect, including RI2474, were selected in order to improve their efficacy by optimizing the dose, the formulation and application conditions. In addition, cytological, biochemical and molecular approaches will be set up to identify the plant defense mechanisms involved in the protection conferred by the three selected RIs.

### Switchgrass Extractives Inhibit Plant Pathogenic Fungi

A. BRUCE (1), B. Ownley (1), J. Tao (1), N. Labbe (1), K. Gwinn (1), D. D'Souza (1), N. Moustaid-Moussa (2)

(1) University of Tennessee, U.S.A.; (2) Texas Tech University, U.S.A.

Switchgrass is a leading contender for biofuel production. Cellulosic ethanol yield is reduced by a phenolic-rich, nonstructural portion of switchgrass, called extractives. Switchgrass extractives hinder enzyme activity and microorganism growth utilized during the biofuel conversion process. Switchgrass would be a more cost-effective feedstock if the extractives portion was developed for use as a biopesticide. The aim of this study was to assess different concentrations of switchgrass extracts for inhibition of *Fusarium oxysporum*, *Fusarium graminearum*, *Alternaria alternata*, *Botrytis cinerea*, and *Bipolaris oryzae*. Crude ethanol extracts were evaluated for inhibition of fungal plant pathogens with disk diffusion and spore germination assays. Colony growth was measured in disk diffusion assays on water agar; treatments were disks treated with 60X and 75X concentrations of extracts, and a 'no extracts' control. Inhibition of mycelial growth was greatest in disk diffusion assays with *A. alternata* and *F. graminearum* at 75X, and *F. oxysporum* at 60X. Spore germination was tested by incubating spores in a water control, or in extracts concentrated at 10X and 15X for 24 h, followed by repeated sampling over a 4-hour period. Germ tube growth was observed microscopically. Spore germination of all fungi was significantly inhibited by both extracts concentrations at all time points. Results indicate that switchgrass extracts have biopesticide potential.

### Quantitative assessment of the efficacy of ZnO nanoparticles against selected fruit fungal contaminants

D. SARDELLA (1), R. Gatt (2), S. Decelis (3), V. Valdramidis (4)

(1) University of Malta, Malta; (2) University of Malta, Metamaterials Unit, Malta; (3) Ministry for Health, Malta; (4) University of Malta, Faculty of Health Sciences, Malta

The use of traditional antifungals to control postharvest diseases has been reviewed or banned during the past years. Nanoparticles represent an alternative for postharvest disease management, either as coating agents or embedded into warehouses' filters. The efficacy of zinc oxide (ZnO) was assessed following the identification of the optimal growth temperature of *Penicillium expansum*, *Alternaria alternata*, *Botrytis cinerea* and *Rhizopus stolonifer*. Fungi were inoculated (10⁵-10⁶ spores/mL) onto SDA plates and incubated at temperatures from 5°C to 35°C under constant *a*<sub>w</sub>. Mycelium diameters were measured and then fitted to a linear model to estimate the growth rate (*μ*) and the apparent lag time (*λ*). The properties of ZnO (3 to 15 mM) were evaluated for the previously identified optimal growth temperature by mycelium diameter and optical density (OD) measurements recorded every 20 min for up to 6 days with a 96-well microtrotile reader at 600 nm. *P. expansum* and *B. cinerea* could grow even at 5°C while *R. stolonifer* had the highest *μ* = 0.383 ± 0.002 (cm/h) at 25°C and the shortest *λ* = 17.703 ± 0.228 (h) at 18°C. The antifungal tests showed *R. stolonifer* and *P. expansum* as the most sensitive to ZnO being completely inhibited even at 3 mM. *A. alternata* and *B. cinerea* were inhibited at 12-15 mM. These results were confirmed by the OD assay. ZnO nanoparticles thus appear as an effective antifungal and their efficacy can be assessed by quantitative mycological studies.

### Recommendation of Clove oil to minimize Penicillium based postharvest losses in pomegranates (Punica granatum L.)

M. ALAM (1), M. Alam (2), A. Rehman (3), M. Amin (4), K. Riaz (3)

(1) Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan, Faisalabad, Pakistan; (2) Department of Plant Pathology, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan; (3) Department of Plant Pathology, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan; (4) Institute of Horticultural Sciences, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan

Plant essential oils are important alternate to synthetic fungicides for the management of postharvest diseases in fruits. In the present study, among the tested clove (*Syzygium aromaticum*), coriander (*Coriandrum sativum*) and oregano (*Oreganum vulgare*) essential oils, clove oil not only inhibited the mycelial growth of *Penicillium expansum* and *Penicillium implicatum* at the tested concentrations (25, 50, 100 and 250 µl/L, performing maximum at 250 µl/L giving 100% fungal growth inhibition), but also showed 64 and 45% disease reduction against *P. expansum* and *P. implicatum* respectively, when applied as curative treatment to the artificially inoculated fruits (1 × 10⁶ spores/mL). In addition, Clove oil treatment also reduced weight loss and maintained soluble solid contents, titratable acidity and ascorbic acid contents of pomegranate fruits for about 4 weeks at ambient temperature (25°C). Hence, it is concluded that clove oil can be used for postharvest disease management, preservation and extension of shelf life of pomegranate fruits.

### Summary of 11-year field trials of using Heads Up® - a Saponin plant protectant to manage soybean diseases

S. Navi (1), X. Yang (1)

(1) Iowa State University, U.S.A.

Heads Up® is a plant protectant that induces systemic acquired resistance of plants when applied as seed treatments or foliar sprays. Since 2005, field trials have been established in multi-locations of Iowa State University Research Farms to determine effectiveness of seed treatment to manage soybean
sudden death syndrome and white mold. Eleven-year Iowa data together with some data from other mid-west soybean productions states were pooled and analyzed. Results show that in average seed treatments with Heads Up alone or Heads Up plus other compound improved soybean yields 2.08 bushels per acre over untreated control. Seed treatments effectively reduced or suppressed the occurrence of SDS and white mold in soybean. Compared with control, the probabilities of yield response to a treatment were 63.5%, 7%, 29.5% for increase, no change, and decrease, respectively. Compared with synthetic chemicals, there is a slightly larger number of low responsive trials for Heads Up, which is often seen in field trials with biological control products. However, the low cost ($<2 per acre) and its biological nature makes Heads Up a good candidate for soybean disease management. To improve consistency, factors leading to low responsiveness are investigated and addressed.

## Efficacy of synthetic and biorational products against powdery mildew of flowering dogwood

P. LIYANAPATHIRANAGE (1), T. Simmons (1), M. Kabir (1), K. Addesso (1), F. Baysal-Gurel (1)

(1) Tennessee State University, U.S.A.

Flowering dogwood (Cornus florida L.) is a popular ornamental plant in home gardens and commercial settings. Powdery mildew, caused by *Erysiphe pulchra*, may cause cosmetic damage on flowering dogwood by attacking tender shoots and leaf surfaces that cause for dead patches and premature defoliation or kill seedlings. Various products are available or in development that have the potential to contribute to the management of powdery mildew of dogwood. In this study the effect of biorational products (Triact 70-neem oil, thymol and cedar oil) and fungicide (Cleary's 3336F) on dogwood powdery mildew severity was evaluated. Treatments were applied using hand-held sprayer every 7 days for a total of five applications. The experiments were done twice, each with three replicates per treatment. The severity of powdery mildew on foliage and phytotoxicity were evaluated using a scale of 0-100% foliage affected. In both experiments, the severity of powdery mildew was low to moderate (~15 and 30% foliage affected-non-treated control). Triact 70, thymol and Cleary's 3336F provided significant control of powdery mildew compared to the non-treated control in both experiments. Powdery mildew disease progress was lower in dogwood plants treated with Triact 70, thymol, Cleary's 3336F and the high rate of cedar oil than in those non-treated control and the low rate of cedar oil. However, thymol and the high rate of cedar oil were slightly phytotoxic on dogwood foliage.

## Antifungal effect of nano sized zero valent iron (nZVI) to special fungal pathogens in paprika and tomato crop

M. PARK (1), J. Kim (1), Y. Lee (2), S. Kim (2)

(1) Cheorwon Plasma Research Institute, South Korea; (2) Department of Applied Plant Sciences College of Agriculture and Life Sciences/Gangwon National Univ., South Korea

Zero-valent iron (ZVI) has been utilized in environmental remediation including the removal of pollution or contaminants such as heavy metals, phosphate as an ingredient of fertilizers and pesticides in groundwater, soil and/or sediment. Recently, many studies have shown that the nano-sized ZVI (nZVI) have antibacterial and antifungal activity by oxidative reaction of the nZVI. Here, we are trying to apply the ZVI or nZVI as antifungal agent instead biocidal chemical compound (fungicide) for *Collectorichum acutatum*, *Fusarium oxysporum* f. sp. *Lycopersici*, *Sclerotinia minor*, *S. sclerotiorum*, and *Rhizoctonia solina*, the causal agent of major fungal disease of paprika and tomato crop in Korea. The result of antifungal activity tests showed that ZVI and nZVI were the most efficient antifungal agent to all five fungal pathogens. Especially, nZVI was more efficient to inhibit the hyphal growth than ZVI in five fungal pathogens.

## Natural products reduce grey mold of tomato caused by Botrytis cinerea

F. AHMED (1), A. Alvarez (1), B. Sipes (1)

(1) University of Hawaii at Manoa, U.S.A.

Grey mold is the most important postharvest disease of tomatoes in Hawaii. Treatments with edible, natural products are needed to reduce losses and contribute to food sustainability. Based on the hypothesis that inhibition of Botrytis spore germination will significantly reduce postharvest losses, botanicals were tested for their effects on conidia. Ten Botrytis isolates from rotting tomato fruit were identified morphologically and confirmed by DNA sequence analysis. Leaves of candidate plants were frozen at -20°C° and plant fluids were sterilized by passing through a 0.22 µm millipore. Extracts were evaluated by adding Botrytis spores (10⁶/ ml) with sterilized malt extract broth to multi-well microplates. Changes in optical density measured at 6, 12, and 24 hours after inoculation were analyzed with Gen5 software. Capsicum chinense cultivars Datil and C. annuum Carnival completely inhibited fungal germination in preparations containing 30 and 40% leaf extract at all evaluation times. These extracts were superior to all other extracts tested. Extracts of Waltheria indica and C. frutescens had intermediate antifungal activity. Aloe vera, Tagetes patula and Capsicum annuum cultivars Red pepper, Criolla de cocina, New Mexico, showed no measurable antifungal activity. Extracts from the two most promising pepper cultivars, Datil and Carnival, are being evaluated as pre-harvest sprays in the greenhouse and as edible coatings on tomato fruit postharvest to reduce grey mold.

## Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita

J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2)

(1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China

Chemical pesticide is an effective way in controlling tobacco disease, but it caused some problems such as environment pollution, ecological balance destruction and chemical residues. So biology control of tobacco disease attracts more and more attention. The usage of micro-organisms or microbial metabolites is an important approach in controlling nematodes. Sphingobacterium nematocida ZY-71-1 is a bacterial endophyte, was isolated from tobacco leaves, which producing volatiles with nematicidal activities. The biocontrol effect of the bacterium towards Meloidogyne incognita was evaluated under Petri dish test and greenhouse assay. The mortality rate to the nematode caused by S. nematocida ZY-71-1 was 100% and the inhibition rate to the hatch of the eggs of the nematode was also 100%. Additionally, nematicidal bioactivity materials produced by S. nematocida ZY-71-1 were determined. By GC/MS analysis and pure chemicals confirmation, 5 volatiles with nematicidal activities against juveniles and eggs of M. incognita were identified from S. nematocida ZY-71-1, including benzeneacetaldehyde, 2-nonanone, decanal, 2-undecanone and dimethyl disulfide. This study provided an effective way for biological control of nematode and established foundation on controlling the nematode.

## The Role of the Citrus Microbiome in Tree Health and Tolerance to Pathogens

N. GINNAN (1), T. Dang (2), P. Ruegger (2), J. Borneman (2), P. Rolshausen (2), G. Vidalakis (2), S. Bodaghi (2), M. Roper (2)

(1) University of California Riverside - Department of Plant Pathology and Microbiology, Riverside, CA, U.S.A.; (2) University of California Riverside, U.S.A.

There is compelling evidence that the health of an animal or plant is linked to the structure and activities of its associated microbiome. The goal of this project is to identify naturally occurring microbes or consortia of microbes that either directly or indirectly protect citrus trees from plant pathogens. We have, thus far, isolated and identified the culturable microbes of the roots, leaves, and budwood of citrus trees collected from California and Florida. We isolated both fungi and bacteria, and sequenced the amplified 16S rRNA gene from bacteria and the ITS region of the ribosomal operon from fungi for microbial identification. A culture-independent analysis of the citrus microbiome using an Illumina MiSeq platform has also allowed us to reveal a more comprehensive view of the microbial communities that inhabit citrus. We hypothesize that the citrus microbiome and their associated metabolomes can be used to reduce or prevent colonization of citrus pathogens, such as *Candidatus* Liberibacter asiaticus (CLas). We are currently screening compounds produced by these isolated citrus microbes for inhibitory properties against CLas. We envision the discovery of bactericide producing microbes that
could be directly transferred and established on trees, or the naturally produced bactericide could be purified and applied to combat Clas and/or other pathogens.

## Fungal antagonism of bacteria isolated from Cultivated and Wild Cranberry Bogs

S. SOBY (1), G. Ebadzad (1), E. Batory (1)

(1) Midwestern University, U.S.A.

Natural environments are characterized by a relative equilibrium that can be brought into disequilibrium by the introduction of agricultural plants. Both natural and agronomic systems should thus contain microbes that contribute to system equilibria by suppressing parasitic organisms. However, few biological niches have been systematically explored for biological control agents. Cranberry bogs in southeastern Massachusetts are a good model system for these studies because of the genetic similarity between wild and domesticated cranberry plants, and their location in the same geographic region. Soil and plant samples were collected from wild and cultivated bogs over several growing seasons to determine the microbial composition of cranberry bogs. 68 bacterial isolates were assessed for *in vitro* inhibition of the pathogenic fungi, *Phomopsis* sp., *Colletotrichum* sp., and *Coleophoma* sp. isolated from cranberry, as well as *Trichoderma* sp. Individual isolates varied in antifungal ability within and between fungi. *Bacillus* spp. were most antagonistic to these fungi, but several other bacterial genera were active against one or more of the fungi as well. In a preliminary assessment, 82% of the chosen bacterial isolates had activity against *Phomopsis*, 44% against *Colletotrichum*, 63% against *Coleophoma*, and 19% against *Trichoderma*. These results suggest that bog soils and plants harbor a large number of candidates for development as biological control agents in agriculture.

## Relative efficacy of Zambian atoxigenic *Aspergillus flavus* strains against aflatoxin-producing *Aspergillus* species

J. AKELLO (1), T. Dubois (2), J. Atehnkeng (3), M. Mukanga (4), H. Njapau (5), J. Augusto (6), P. Cotty (7), R. Bandyopadhyay (3)

(1) International Institute of Tropical Agriculture (IITA-Zambia), Zambia; (2) The World Vegetable Center (AVRDC) Eastern and Southern Africa, Arusha, Tanzania; (3) International Institute of Tropical Agriculture (IITA-Nigeria), Ibadan, Nigeria; (4) Zambia Agricultural Research Institute, Mount Makulu Central Research Station, Lusaka, Zambia; (5) National Institute for Scientific and Industrial Research (NISIR), Lusaka, Zambia; (6) International Institute of Tropical Agriculture (IITA-Mozambique), Nampula, Mozambique; (7) USDA-ARS, School of Plant Sciences, University of Arizona, Tucson, U.S.A.

In Zambia, fields cropped to maize and groundnuts are predominantly colonized by aflatoxin producing Aspergillus flavus S-morphotypes and A. parasiticus. Consequently, Zambian staple foods are often contaminated with high aflatoxin levels (>20 ppb) while in the field and during storage. This study aimed at identifying the best atoxigenic A. flavus L-strains for use as biocontrol agents. In vitro, the most toxigenic L (25) and SBG (10) strains were selected out of the entire isolate library based on qualitative and quantitative TLC assessment. Competition assays were performed by co-inoculating 14 atoxigenic candidate strains and the 35 selected toxigenic ones on sterile maize grains in sterile vials. All the 14 atoxigenic strains outcompeted the toxigenic ones and reduced aflatoxin production by >96% in vitro. Eight of the 14 atoxigenic strains were selected and formulated into two mixes, with each mix comprising of 4 strains. Each mix was applied to maize and groundnut fields between 2012 and 2015 at a rate of 10 kg/ha, 2-3 weeks before flowering. Treating fields with the two mixes altered Aspergillus soil community structure and significantly reduced aflatoxin levels in maize and groundnuts by 89.6% compared to untreated ones. Thus, the 8 atoxigenic strains hold promise for fighting aflatoxin burden in maize and groundnut. Adopting such strains to address aflatoxin could improve grain nutritional quality and safety for better health of the Zambian populace.

## Incidence of soybean frogeye leaf spot in Korea and selection for antagonistic bacteria controlling *Cercospora sojina* Hara

I. KANG (1), I. Kang (1), H. Shim (2), D. Shin (2), J. Roh (2), S. Heu (2)

(1) National Institute of Crop Science, South Korea; (2) National Institute of Crop Science, South Korea

Soybean frogeye leaf spot caused by the fungus *Cercospora sojina* Hara, has been seen a steady increase in the incidence and prevalence on soybean in Korea. The nationwide survey of the occurrence of frogeye leaf spot showed that 11 out of 28 regions in 2015 had been infected with *Cercospora sojina* Hara. To control the disease, an isolate which showed strong inhibitory effect on the mycelial growth and conidial germination of *Cercospora sojina* Hara was selected among the antagonistic bacterial isolates collected from soybean grown soil and the bacterial isolate was identified as *Paenibacillus polymyxa* using 16S rRNA sequence analysis. *Bacillus subtilis* QST713(Serenade) was also inhibitory effect on the mycelial growth and conidial germination. The inhibition rates of these antagonistic bacteria were 53%, 75%, respectively. They could be effectively used for controlling soybean frogeye leaf spot.

## Integration between 2,4-DAPG-producing *Pseudomonas* and fluquinconazole to control take-all disease of wheat in Chile

E. MOYA ELIZONDO (1), C. Vera (1), R. Madariaga Burrows (2)

(1) Universidad de Concepción, Chile; (2) Instituto de Investigaciones Agropecuarias, INIA Quilamapu, Chile

Take-all disease of wheat, caused by the fungus *Gaeumannomyces graminis* var. tritici (Ggt) could be controlled by integrating fungicide seed treatments with 2,4-Diacetylphloroglucinol-producing *Pseudomonas* spp. (2,4-DAPG). This research assessed the effect on yield parameters and disease damage associated with the integrated use of seed treatments with two Chilean 2,4-DAPG-producing *Pseudomonas* spp. (10⁶ ufc/g grain seeds) and the triazole fungicide fluquinconazole (1.8 mL/g grain seeds). A field experiment was established in Chillán, Chile, during crop season 2015. Experimental field plots of 2.4 m² with winter wheat cv. Maxwell were seeded to assess combinations with or without the mentioned seed treatments, plus two untreated control (infested or non-infested with Ggt) on a randomized block design. Seed treatments where both strains of *Pseudomonas* were mixed with the fungicide reduced plant and root rot disease severity damage (50 and 49%, respectively) and white heads (32%), while increased yield (34%) and grain quality (2.1%) with respect to the Ggt infested control (P≤0.05). Single use of bacterial seed treatments without the fungicide was not different from this control (P≥0.05). Fluquinconazole alone had a lower grain yield (13%) and wheat test weight (1.4%) than when it was mixed with the two bacterial strains. Results suggest that integration of 2,4-DAPG-producing *Pseudomonas* with fungicides could reduce take-all disease and increase yield in wheat crops.

## Stopping the cereal killer: Exploring biological control to mitigate Fusarium head blight of wheat

A. BEHARI (1), G. Kuldau (1)

(1) Pennsylvania State University, U.S.A.

Fusarium head blight (FHB) is a devastating fungal disease impacting wheat and other cereal production globally. Exacerbating the impact of disease is the *in planta* production of the toxic fungal secondary metabolite and virulence factor, deoxynivalenol (DON). DON is an inhibitor of the eukaryotic ribosome thereby blocking protein translation, and in toxicological studies has been found to affect the gastrointestinal, immune, and endocrine systems of humans and animals. Preventing DON accumulation in grain ultimately begins with preventing infection by FHB causing *Fusarium* species. Current mitigation strategies to combat FHB include the use of moderately resistant varieties of wheat, crop rotations with non-hosts, and fungicides, but even in conjunction these strategies provide only minimal to moderate control when environmental conditions are conducive to disease. This research aims to identify and characterize biological control agents to mitigate FHB through media based screening of samples from different production systems and geographic locations for *Fusarium graminearum* inhibiting and DON degrading microbes. Exploring multiple sources including various soils, plant material, silage, and haylage will allow for a diverse range of microbes exhibiting the aforementioned capabilities to be studied for their specific mechanisms, potential common genetic basis, and potential use in microbial consortia for disease control.
**Characterization and distribution of natural populations of *Cryptococcus flavescens* across the United States**

F. ROTONDO (1), B. McSpadden Gardner (2), P. Paul (1)

(1) The Ohio State University, Wooster, OH, U.S.A.; (2) The Ohio State University, Wooster, U.S.A.

Fusarium head blight (FHB) is an important disease of small grains caused primarily by *Fusarium graminearum*. This disease causes grain yield and quality losses under conducive conditions. The yeast *Cryptococcus flavescens* is an effective biological control agent of FHB on wheat. In this study, the biogeography and genetic diversity of natural populations of *C. flavescens* across the United States were characterized. Wheat spikes samples were collected at flowering and at harvest and assayed for *C. flavescens* using specific PCR primers designed on the heat shock protein *hps70KDa*. This assay is 1,000 times more selective for *C. flavescens* than other *Cryptococcus* spp. Samples with > 106 cells/g of wheat tissue were subjected to microbial isolation, and colonies with *C. flavescens*-like morphology were selected and characterized using amplified ribosomal DNA restriction analysis (ARDRA) profiling and multilocus genotyping. The results showed that *C. flavescens* was widespread across the country, albeit at low incidence (approximately 10%, n = 320). Spikes were the preferred tissue of colonization. When present, populations of *C. flavescens* ranged from 105-107 cells/g of tissue. Plant growth stage influenced *C. flavescens* colonization. This work confirms that *C. flavescens* is found across a broad geographical area in close association with wheat. Further, it demonstrates that *C. flavescens* is a non-invasive biological control option for FHB in the regions sampled.

**Biocontrol of Fusarium head blight with *Bacillus* spp.**

K. BOWEN (1), J. Anderson (1), K. Liu (1)

(1) Auburn University, U.S.A.

Fusarium head blight, caused by *Fusarium graminearum*, has become a cause of concern in many portions of Alabama. This disease was severe in Southern AL, due to frequent rainfall during flowering in 2015. Infection of *F. graminearum* reduces quantity and quality of yield by infecting the kernels of many grain crops; it also produces the mycotoxin, deoxynivalenol (DON). Wheat grain was collected from fungicide treated plots and found to be infested with *Fusarium* spp. Cultures were established from single spores and species were determined from morphology of spores and color of the fungus when mature. Biocontrol potential of selected *Bacillus* strains against *F. graminearum* was assayed using a dual culture method. Based on inhibition of fungal growth and spore production, strains were selected that were most suppressive against *F. graminearum*. In order to test the effectiveness of these strains in the field, five *Bacillus* strains will be applied to wheat heads during flower, and disease intensity will be rated 3 weeks later. Results of the field study will be reported. Any reduction in Fusarium head blight by a biological agent might contribute to overall management of this disease.

**Selection and characterization of probiotics for rye silage**

C. JEON (1), H. Kim (1), J. Kim (1), Y. Kwak (2)

(1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea

Silage is feed of livestock. Forage crops are at risk from infection by mycotoxins producing fungi. Rye has considerate as a highly quality of forage. After heading stage, the biomass of rye increased up to 30%. However, lignification caused low fermentation efficiency and coefficient digestibility. This study was performed to develop probiotics which improve quality of silage in nutrient aspect. During the fermentation period of the rye silage, dominant microorganisms were selected. Those were confirmed antimicrobial activities against fungi and were tested acidification ability. The isolates confirmed ability of fibrinolysis through the four enzyme (cellulase, xylanase, esterase, chitinase). The final selected strains were added to the rye silage and then confirmed the effect by real-time PCR in antifungal activity. Total 180 isolates were isolated from the rye silages during various fermentation period. Among 180, 8 isolates showed antimicrobial activities against *Fusarium moniliforme*. two isolates among antimicrobial activities isolates showed strong acidification ability. Also a single isolate was selected that was effective all four enzymes. In real-time PCR results, the mycotoxin gene was detected significantly low in the rye silage, which added antimicrobial activity isolate. In case developed microorganism product was applied at other stock feed as well as rye silage, production of high quality forage will expand.

**Inhibition of *Penicillium expansum* growth on Golden Delicious apples by wild yeasts from various plant materials**

V. TOURNAS (1), E. Katsoudas (2)

(1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/ORA/FDA, U.S.A.

Wild yeast strains previously isolated from various fruits were tested for their ability to inhibit *Penicillium expansum* in vivo (on Golden Delicious apples) in an effort to determine their potential as biocontrol agents (BCAs). Tests were performed at room temperature (~21°C) for up to 2 weeks and at refrigeration (3°C) for up to 2 months. *Candida oleophila* was used as positive BCA control. All strains tested showed some degree of biocontrol activity against *P. expansum*. At room temperature, activity (lesion size reduction) ranged between 37 and 50% during the first week. After 14 days of incubation, decays on yeast-treated apples were slightly smaller (16-19%) than those on apples treated with *P. expansum* alone. At refrigeration, lesion reduction ranged between 54 and 76% in the first month of storage and between 60 and 76% after 2 months of incubation. Decay incidence was 100% in apples stored at room temperature and 25-62% in those kept under refrigeration. The inhibitory activities of the wild yeasts were similar to those exhibited by *C. oleophila*. These strains showed potential as BCAs against *P. expansum* on stored apples.

**Evaluation of potential biocontrol agents against fungal plant pathogens affecting strawberry**

R. BITTER (1), G. Holmes (2)

(1) California Polytechnic State University, U.S.A.; (2) Cal Poly Strawberry Center, U.S.A.

*Botrytis cinerea*, *Fusarium oxysporum* f. sp. *fragariae* and *Macrophomina phaseolina* are important pathogens that limit commercial strawberry production in California. The objective of this study was to evaluate eight species of bacteria (19 strains) for their ability to inhibit mycelial growth of all three pathogens *in vitro*. Percent inhibition of fungal growth due to the presence of each bacterium was determined relative to growth in the absence of each bacterium. Three strains of *Bacillus amyloliquefaciens* provided the greatest inhibition of *B. cinerea*, *F. oxysporum* f. sp. *fragariae* and *M. phaseolina* by an average of 43.7%, 47.8%, 42.7%, respectively, between replications. Significant differences in fungal inhibition were observed between strains of the same bacterial species, suggesting inhibition is strain-dependent. Growth inhibition caused by five *B. subtilis* strains varied from 39.5% to 0.6% for *B. cinerea*, 43.1% to 0.6% for *F. oxysporum* f. sp. *fragariae*, and 39.8% to 5.8% for *M. phaseolina*. Four *B. licheniformis* strains also displayed similar levels of strain-dependent inhibition. Bacterial antagonists that limit the growth of these soilborne and foliar pathogens could provide an alternative to the use of chemical fumigants and fungicides, or could be utilized to enhance the effectiveness of conventional or organic disease control treatments such as anaerobic soil disinfestation.

**Effects of *Clonostachys* species on charcoal rot disease caused by *Macrophomina phaseolina* in soybean**

A. ADESEMOYE (1), H. Wei (1)

(1) University of Nebraska Lincoln, U.S.A.

Charcoal rot disease caused by *Macrophomina phaseolina* in soybean greatly disrupt stand establishment and cause high yield losses. The pathogen was estimated to cause yield losses of over 270,000 bushels in Nebraska (NE) in 2011. Chemicals are used against many diseases but not consistently effective in the management of soilborne diseases such as charcoal rot. In 2014 and 2015, surveys were conducted in soybean fields in NE. Infected
samples were collected, plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated in the dark for 4 days at 25°C. Morphology was examined and genomic DNA was extracted from isolates using MoBio microbial isolation kits. Two genes of interest, ITS and β-tubulin, were PCR-amplified. Amplicons were sequenced at the University of California Riverside Gencore. The pathogen *M. phaseolina* was identified but different species of *Clonostachys* (anamorph: *Bionectria* spp.), including isolates identical to *C. rhizophaga*, *C. byssicola*, and *C. rosea* were also recovered from the samples. It appeared from the field samples that *Clonostachys* spp. had negative impacts on the population of *M. phaseolina*. In repeated *in vitro* assay as well as *in vivo* co-inoculation through Rolled Towel Assays indicated suppression of *M. phaseolina* by *Clonostachys* spp. There is need for further studies to better understand the biocontrol efficacy of each *Clonostachys* sp. as well as the mechanisms involved.

## Trichoderma asperellum survival and parasitism of *Sclerotinia sclerotiorum* sclerotia in different soil types

M. LOBO (1), A. Geraldine (2), F. Yoshida (3), E. Civardi (3), E. Barbosa (1)

(1) Embrapa (Brazilian Agricultural Research Corporation), National Center for Rice and Beans Research, Santo Antônio de Goiás, Brazil; (2) Instituto Federal Goiano, Rio Verde, Brazil; (3) Universidade Federal de Goiás, Goiânia, Brazil

Effectiveness of bioagent *Trichoderma asperellum* is influenced by physical, chemical, biological and environmental factors, but there is no clear idea of how such variables affect antagonist endurance. This study assessed the survival of *T. asperellum* and the parasitism of *Sclerotinia sclerotiorum* sclerotia in different Brazilian soils. The test was carried out in a completely randomized design, arranged in a 4 × 6 factorial schedule with five repetitions. Experimental factors comprised six soils of distinct texture and fertility profiles, and four biocontrol treatments with three *T. asperellum* isolates and a water control. Plots consisted of 12cm x 22cm x 3cm transparent plastic containers, with sclerotia distributed over 300g of soil. After spraying with 2.0 × 10¹² *T. asperellum* conidia ha⁻¹ and soil moisture adjusted to 100% field capacity, treatments were incubated at 20°C with 12h photoperiod for 28 days. Weakly assessments considered sclerotia parasitism, area under antagonist survival curve (AUASC), culturable fungi and bacteria, and bacterial diversity estimated with Biolog Ecoplates. Principal component analysis showed that parasitism of *S. sclerotiorum* sclerotia was proportional to *T. asperellum* AUASC. In contrast, antagonist survival was negatively affected by soil pH, bacterial density and diversity, organic matter, Ca, Mg and K contents. Biocontrol efficiency varied according to soil source.

## In-vitro control of *Calonectria pseudonaviculata* by bacteria recovered from irrigation water

X. YANG (1), C. Hong (2)

(1) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A.; (2) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A.

*Calonectria pseudonaviculata* (*Cps*) is the causal agent of boxwood blight. This disease first observed in USA in 2011 has significantly impacted the nursery and landscape industries. The objective of this study was to develop biologically-based, long-term solutions to managing this emerging disease at production facilities and in the landscapes. Over 1000 bacterial strains recovered from irrigation water in Virginia, USA were screened for their potential as biological control agents against microsclerotia of *Cps*. Microsclerotia were harvested from potato dextrose agar cultures and plated on nutrient agar in 48-well plates. Two days later, fresh bacterial cultures grown in nutrient broth were added to the culture of *Cps*. Three replicate wells per bacterial strain were used. Sterile nutrient broth was added to three wells per plate as controls. Microsclerotia germination and resultant mycelial growth was measured based on a 0-to-5 rating scale at five and ten days after adding bacterial cultures. It was found that approximately 30% of screened bacterial strains greatly suppressed microsclerotial germination and mycelial growth with average ratings of one or less. These bacteria are promising biological control agents (BCAs) for boxwood blight and warrant further evaluation using detached leaf assay, greenhouse and field *in-vivo* tests.

## Characterization and distribution of natural populations of *Cryptococcus flavescens* across the United States

K. PARK (1), J. Shin (1), K. Myeong (1), S. Lee (1), S. Lee (1), M. Kim (1), K. Kim (1)

(1) National Institute of Horticultural and Herbal Science, South Korea

Fusarium head blight (FHB) is an important disease of small grains caused primarily by Fusarium graminearum. This disease causes grain yield and quality losses under conducive conditions. The yeast *Cryptococcus flavescens* is an effective biological control agent of FHB on wheat. In this study, the biogeography and genetic diversity of natural populations of *C. flavescens* across the United States were characterized. Wheat spikes samples were collected at flowering and at harvest and assayed for *C. flavescens* using specific PCR primers designed on the heat shock protein hps70KDa. This assay is 1,000 times more selective for *C. flavescens* than other *Cryptococcus* spp. Samples with > 106 cells/g of wheat tissue were subjected to microbial isolation, and colonies with *C. flavescens*-like morphology were selected and characterized using amplified ribosomal DNA restriction analysis (ARDRA) profiling and multilocus genotyping. The results showed that *C. flavescens* was widespread across the country, albeit at low incidence (approximately 10%, n = 320). Spikes were the preferred tissue of colonization. When present, populations of *C. flavescens* ranged from 105-107 cells/g of tissue. Plant growth stage influenced *C. flavescens* colonization. This work confirms that *C. flavescens* is found across a broad geographical area in close association with wheat. Further, it demonstrates that *C. flavescens* is a non-invasive biological control option for FHB in the regions sampled.

## Curvularia prsadii as a biological control agent for barnyard grass (*Echinochloa crus-galli*) in rice fields

Y. SHABANA (1), A. Abu Tabl (1)

(1) Mansoura University, Egypt

*Echinochloa crus-galli* is one of the most important damaging weeds of rice paddies. Three isolates of *Curvularia prasadii* (CP01, CP02, and CP03) were isolated from the rice weed, *E. crus-galli* in three governorates (Dakahlia, Kafr El-Sheik, and El-Garbeia) in Egypt in three consecutive growing seasons (from 2012 to 2014). The biocontrol activity of these fungal isolates was determined on the weed seedlings in the greenhouse using spore suspension of 10⁶ conidia/ml. All of the three isolates tested produced 40-60% disease severity on the weed seedlings with no significant differences among them. Host range studies were conducted to determine the host specificity and safety of the three isolates of the bio agent, *C. prasadii* against 23 non-target plant species and cultivars of economic importance grown in Egypt. No disease symptoms appeared on any of the non-target plants tested. The optimum nutritional and physical conditions for the production of the biocontrol agents were determined and will be discussed.

## Biocontrol of Tubakia leaf blight by *Paenibacillus peoriae* (HB774)

H. YUN (1), Y. Kim (1)

(1) Seoul National University, South Korea

South Korea has shown decreasing proportion of coniferous plants and increasing proportion of deciduous plants due to the effect of global warming. With the growing significance of deciduous forests, the national forest damage by ongoing Tubakia leaf blight of the oak is more severe than before. In response to the current critical forest situation of Korea, this study aims to develop the efficient biocontrol agent of the Tubakia leaf blight. Among 30 bacteria isolated from soil and plants, HB774 showed the highest inhibition rate (85.8%) of the fungal mycelial growth, which was selected as a potentially effective biocontrol agent. As a result of the 16S ribosomal DNA sequence analysis, a gram-positive bacterial isolate was identified as *Paenibacillus peoriae* and deposited as a patent strain in Rural Development Administration of Korea. This isolate inhibited the mycelial growth by 69.7% and 75.6% at the bacterial concentrations of 1 × 10⁶ CFU/ml and 1 × 10⁸ CFU/ml, respectively, of which the inhibitory rates were identical to or significantly higher than those of the fungicides, propiconazole and iminonctadine-triacetate. In the simultaneous treatment and pre-treatment of HB774, its control efficacies were high with 80-90%, showing no significant differences from those of propiconazole and iminonctadine-triacetate. This suggests *P. peoriae* HB774 has a high potential to be developed as an efficient biocontrol agent against the Tubakia leaf blight on oaks.
### Acaromyces ingoldii inhibits the laurel wilt pathogen, *Raffaelea lauricola* in vitro

R. OLATINWO (1), S. Fraedrich (2)

(1) USDA Forest Service, Southern Research Station, U.S.A.; (2) USDA Forest Service, Southern Research Station, U.S.A.

Laurel Wilt is a destructive disease of redbay (*Persea borbonia* and other species in the laurel family (Lauraceae) in the southeastern United States (US)). The disease is caused by *Raffaelea lauricola*, a fungal symbiont of the redbay ambrosia beetle (*Xyleborus glabratus*). The beetle and the fungus were introduced into the US probably with wood packing materials. This investigation was prompted by antifungal activities demonstrated by an *Acaromyces* species isolated from a loblolly pine bolt in Louisiana during routine laboratory microbial isolations. Our objective was to evaluate the inhibitory property of the *Acaromyces* species against *R. lauricola*. We obtained isolates of *R. lauricola* from fresh laurel wilt infected tissues collected from sassafras trees in Louisiana and Texas, and evaluated the competitive ability of the *Acaromyces* and the fungistatic activities of secondary metabolites produced by the fungus against *R. lauricola* in vitro. Results showed a strong inhibition of *R. lauricola* mycelia growth by the *Acaromyces* species. Growth inhibition was significant as pigmented secondary metabolites produced by *Acaromyces* diffuses through the media. Only minimal direct competition was observed between the two fungi. *R. lauricola* isolates inoculated 7-day after *Acaromyces*, showed no growth and no spore germination. Future experiments will examine the identity, efficacy, and potential benefits of secondary metabolite(s) produced by the *Acaromyces* species.

### Applications of *Bacillus amyloliquefaciens* PMB01 for managing Fusarium wilt of cucumber

W. DENG (1), H. Chou (2), I. Yang (3), Y. Lin (4), H. Wang (2), S. Chuang (2), T. Huang (2)

(1) National Chung Hsing University, Taichung, Taiwan; (2) Kaohsiung District Agricultural Research and Extension Station, Pingtung, Taiwan; (3) Taiwan Agricultural Mechanization Research and Development Center, Taipei, Taiwan; (4) National Pingtung University of Science and Technology, Pingtung, Taiwan

Cucumber Fusarium wilt, caused by *Fusarium oxysporum* f. sp. *cucumerinum* (*Foc*), is the most devastating disease on cucumber. Currently, no effective chemicals are available for disease control; however, prior studies showed that antagonistic rhizobacteria can reduce the disease severity, suggesting that biocontrol agents might be potential solutions for the management of Fusarium wilt disease. In this study, soil bacteria with inhibitory activity on in-vitro *Foc* growth were isolated. Among them, *Bacillus amyloliquefaciens* PMB01 showed the best antagonistic activity against *Foc* in confrontation assays. The efficacy of PMB01 on controlling Fusarium wilt disease of cucumber (FWC) was evaluated in field experiments, and the results showed that applications of the formulated PMB01 reduced the disease severity of FWC down to less than 15%. Prior research also revealed that the formulated PMB01 effectively controlled bacterial wilt disease of tomato (BWT), caused by *Ralstonia solanacearum*, in field trials. Whole genome shotgun sequencing showed that PMB01 harbors genes involved in the biosynthesis of cyclic lipopeptides, bacillibactin, and bacilysin, which might collectively contribute to its biocontrol activity. Toxicology testing demonstrated that PMB01 was non-toxic to mammals. In 2016, the biopesticide Saviour® containing *B. amyloliquefaciens* PMB01 as the active ingredient was developed and commercialized in Taiwan for integrated management of BWT and FWC.

### The potential use of a *Cladosporium* sp. as a biological control agent for white mold caused by *Sclerotinia sclerotiorum*

B. ROBINSON (1), R. Cating (2), K. Frost (2)

(1) Oregon State University; Hermiston High School, U.S.A.; (2) Oregon State University, U.S.A.

*Sclerotinia scelortiorum* (Lib.) de Bary is a pathogenic ascomycete known to cause disease in more than 400 plant species, including white mold in potato. In this study, a fungal contaminant found associated with *S. sclerotiorum* that appeared to inhibit *S. scelortiorum* growth was isolated, sub-cultured, and identified. Morphological characteristics and sequence data from internal transcribed spacers 1 and 2, 5.8s rDNA, and *Actin* and *Calmodulin* genes were consistent with the unknown fungus belonging to the *Cladisporium* genus. A bioassay was used to examine inhibition of *S. sclerotiorum* growth by the *Cladisporium* isolate. A 5mm plug of the *Cladisporium* isolate was plated on potato dextrose agar and left to grow for 72 hours before a 5mm plug of *S. sclerotiorum* was plated 50mm from the *Cladisporium* plug. Isolates of *S. sclerotiorum* and *Cladisporium* were plated alone as controls. The plates (n=10 per treatment) were incubated for 4 days at room temperature with a 12:12 photoperiod. Radial growth of *S. sclerotiorum* mycelia was measured and the experiment was repeated in entirety one time. Averaging over both experiments, the radii of *S. sclerotiorum* plated with the unknown isolate was reduced by 34.1 mm (Paired t-test: t = 27.4, df = 19, p < 0.001) when compared to the radii of the *S. sclerotiorum* plated alone. These data indicate that the *Cladisporium* isolate has potential use as a bio-control agent against *S. sclerotiorum* and should be further evaluated in vivo.

### Infection duration of entomopathogenic fungi *Aspergillus* spp. and *Fusarium* spp. against *Bemisia tabaci*

W. Anwar (1), H. Mushtaq (1), M. Haider (1), A. Shahid (1), U. Hameed (1), M. Ur-Rehman (1)

(1) Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, 54590, Pakistan

Entomopathogenic fungi are emerging as biocontrol of insects and new fungi are isolated from different insects with passage of time. The aim of present study was to observe the steps of infection of different *Aspergillus* and *Fusarium* species and their duration of infection against *Bemisia tabaci*. To observe the infection duration, spore suspension of different species of *Aspergillus* and *Fusarium* already isolated from insects were used on nymphal stage of whitefly and data regarding duration of spore germination, penetration, colonization and infection was determined under control conditions. It was observed that spores of *Aspergillus* species germinate quickly as compare to *Fusarium* species. There was no significant difference in spore germination on species level. Penetration time of *Aspergillus* and *Fusarium* species vary and colonization and infection duration of *Aspergillus* species was less as compared to *Fusarium* species against nymphal stage of whitefly. In this study, it was observed that *Aspergillus* is more aggressive as compare to *Fusarium* species and also dependent on isolates of insect associated fungi. Further studies required on genetic level that makes entomopathogenic fungi more aggressive on different stages of infection process.

### Field Performance of Endophytic Actinomycetes in Relation to Plant Growth Promotion and Biological Control of *Fusarium oxysporum* a Pathogen of Tomato

K. EL-TARABILY (1), A. Alkhajeh (1)

(1) United Arab Emirates University, UAE

Four endophytic actinomycete strains identified as *Actinoplanes missouriensis*, *Streptomyces olivaceiscleroticus*, *S. globosus* and *Dactylosporangium aurantiacum* were obtained in a previous study from surface-disinfested tomato roots. The performance of these four strains was evaluated under field conditions to determine their potential to promote plant growth and to reduce the incidence of wilt disease of tomato caused by *Fusarium oxysporum f.sp. lycopersici*. When applied individually or in combination to tomato seedlings, the four strains significantly promoted growth and reduced the incidence of the disease. Individually the performance level of *A. missouriensis* was relatively the best followed by *S. olivaceiscleroticus*, then by *S. globosus* and then by *D. aurantiacum*. The combination of the four strains resulted in significantly better suppression of the disease and growth promotion, compared to individual strains. The ACC deaminase-producing isolates (ACC⁺) (*A. missouriensis* and *S. olivaceiscleroticus*) were significantly more effective in reducing the incidence of the disease compared to ACC deaminase-non-producing (ACC⁻) isolates (*S. globosus* and *D. aurantiacum*). The overall performance of the endophytic strains is promising and could replace the fungicides currently in use for the control of *Fusarium* diseases. The results also showed the potential to enhance the biocontrol agent's performance by including the ACC deaminase ability into the biocontrol strains.
### Assessment of three *Trichoderma* species to protect cabbage from Clubroot

A. BOTERO (1), A. Cotes (2), C. Garcia (3)

(1) Universidad Nacional de Colombia, Colombia; (2) CORPOICA, Colombia; (3) Universidad Nacional de Colombia, Colombia

Following reports that biocontrol agents are potential management tools for clubroot of crucifers, caused by *Plasmodiophora brassicae*, this research aimed at assessing one strain of each of three *Trichoderma* species: *T. asperellum* Th034, *T. brevicompactum* Th201 and *T. koningiopsis* Th003 with different number of applications in terms of disease reduction on 'Delus' cabbage. Experiments were conducted in growth chambers and infested fields. For growth chambers, two concentrations of the pathogen were used, and *Trichoderma* was applied once or twice; disease was measured 60 days after planting. For field experiments *Trichoderma* was applied once, seven or 15 times during the cycle, and evaluations were done quarterly. In growth chambers, disease reduction was observed only for the low pathogen concentration; the highest reduction was observed when *T. koningiopsis* and *T. brevicompactum* were applied once. Disease reduction by *T. asperellum* was proportional to the number of applications. Field results were not as clear cut as those from growth chambers; however, disease reduction was observed with *T. asperellum*, and again this reduction was proportional to the number of applications. In conclusion, *T. asperellum* shows potential for clubroot control when applied frequently, whereas one application of either *T. brevicompactum* or *T. koningiopsis* can be useful in disease management in low pathogen concentrations.

### Evaluation of the biocontrol potential of different *Pseudomonas* species in suppressing Pythium root rot in petunia plugs

D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1)

(1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A.

*Pythium spp.*, causing root rot and damping off, affects 300+ hosts including several ornamental plants, and has developed resistance to two widely used fungicides, mefanoxam and propamocarb. This poses a real threat to greenhouse growers' efficiency in cultivating healthy plants. Ohio State researchers have identified several *Pseudomonas* strains that exhibit biological control potential for disease suppression in many crops, including corn and tomato. One strain of each of the species *P. putida*, *P. brassicacearum*, *P. fluorescens*, *P. poae* and *P. protegens* was tested for the ability to suppress the activity of different *Pythium* species in petunia plugs. Since the production of bacterial metabolites is higher in the transition between logarithmic and stationary phases, our first step included building growth curves of the five strains to identify the appropriate time needed for incubation of the bacterial inoculum prior to application to petunia plugs. Cultures were prepared in LB medium and the OD600 values were recorded using a spectrophotometer every two hours for up to 18 hours. Serial dilution plating at each time point provided correspondent CFUs. 'Carpet White' Petunia seeds were sown in 128-plug trays and immediately treated with a 10⁷ CFU/mL concentration of each *Pseudomonas* strain. After 14 days, plugs were inoculated with the different *Pythium* species. Responses of disease incidence and severity were evaluated until plugs became shipment-ready.

### Microbiome networks: A systems framework for identifying candidate microbial assemblages for disease management

R. POUDEL (1), A. Jumpponen (2), D. Schlatter (3), T. Paulitz (3), B. McSpadden Gardener (4), L. Kinkel (5), K. Garrett (1)

(1) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (2) Division of Biology and Ecological Genomics Institute, Kansas State University, Manhattan, KS, U.S.A.; (3) USDA-ARS, Wheat Health, Genetics, and Quality Research Unit, Washington State University, Pullman, WA, U.S.A.; (4) Department of Plant Pathology, The Ohio State University, Wooster, OH, U.S.A.; (5) Department of Plant Pathology, University of Minnesota, St. Paul, MN, U.S.A.

Network models of soil and plant microbiomes present new opportunities for enhancing disease management, but also challenges for interpretation. We present a framework for interpreting microbiome networks, illustrating how the observed structure of networks can be used to generate testable hypotheses about candidate microbes affecting plant health. The framework includes four types of network analyses. "General network analysis" identifies candidate taxa for maintaining an existing microbial community. "Host-focused analysis" includes a node representing a plant response such as yield, identifying taxa with direct or indirect links to that node, interpreted as either beneficial or detrimental to plant health. "Pathogen-focused analysis" identifies taxa with direct or indirect links to taxon nodes known a priori to represent pathogens, which can be interpreted as agonists and antagonists, respectively. "Disease-focused analysis" identifies key nodes for both plant and pathogen responses. We illustrate the interpretation of network structure with analyses of two microbiomes: the oak phyllosphere and soil associated with the presence or absence of infection by *Rhizoctonia solani*. Such network analyses can be used to further characterize microbial communities and associated conditions involved in the suppression of plant pathogens, the biofertilization of crop plants, and/or the expression of host resistance in crop plants.

### Linking microbial taxa in SDS-suppressive soils of Soybean fields

A. SROUR (1), A. Fakhoury (1), J. Bond (2), L. Leonardo (3), D. Malvick (4)

(1) Southern Illinois University, U.S.A.; (2) Southern Illinois University, U.S.A.; (3) Iowa state University, U.S.A.; (4) University of Minnesota, U.S.A.

Sudden Death Syndrome (SDS) is one of the most important diseases of soybean in the Midwest. SDS is caused by the soilborne fungus *Fusarium virguliforme*. SDS frequently appears in circular to oblong patches in a soybean field and are often found unevenly distributed across the same field, while conditions in the rest of the field may appear to be naturally suppressive to the disease. In the SDS-suppressive areas, soybean plants may be less affected by the pathogen because of the activity of other soil microbes. There is a lack of comprehensive studies that investigate the role of soil inhabiting bacteria and fungi in affecting the development of the SDS. By coupling metabarcoding with paired-end Illumina MiSeq sequencing, we identified key microbial taxa involved in the suppression or promotion of the SDS pathogen. Statistical analyses revealed significant differences in fungal and bacterial community composition between suppressive and conducive soils. A microbial consortia involving members of Actinobacteria, *Fusarium oxysporum*, *Trichoderma*, *Penicillium*, *Metacordycpes*, and *Purpureocillium* were found consistently associated with SDS suppression. In contrast, members of the *Fusarium solani* sp. complex were frequently present in conducive soils.

### Isolation of endophytic bacteria associated with roots from citrus infected with *Phytophthora nicotianae*

A. OLIVARES (1), J. Hernandez-Mendoza (1), S. Nelson (2), V. Ancona (1)

(1) Texas A&M Kingsville Citrus Center, U.S.A.; (2) Texas A&M Kingsville Citrus Center, U.S.A.

Citrus orchards in Texas are greatly affected by *Phytophthora* foot and root rot. Because the presence of antagonistic bacteria associated with plant roots may play a crucial role in root health and disease development we decided to study the effect of *Phytophthora* infection on endophytic bacterial communities associated with citrus roots. Roots from three *Phytophthora* infected and three uninfected grapefruit trees were randomly selected to isolate culturable bacteria. Roots were collected from soil samples taken from each tree at four quadrants, 1 meter away from the trunk at a depth of 15 centimeters. Serial dilutions of macerated root tissues were plated onto nutrient agar, tryptone yeast, King's B and actinomycetes isolation mediums and incubated at three different temperatures. A significantly higher number of cultivable bacteria were present in roots from infected trees versus uninfected trees. In addition, a total of 140 and 119 morphologically distinct bacterial colonies were recovered and isolated from *Phytophthora* infected and uninfected trees respectively. These initial results indicate that citrus trees infected with *Phytophthora* spp. are more prone to develop bacteria-root associations. Characterization of the bacterial species associated with *Phytophthora* infection will lead to the identification of beneficial or antagonistic bacteria against *Phytophthora* spp.

### Seasonal dynamics of fungal communities in pistachio and almond in California: Implications for aflatoxin biocontrol management

A. ORTEGA-BELTRAN (1), R. Puckett (2), T. Michailides (3), D. Morgan (4), J. Moral (3)

(1) UC Davis, U.S.A.; (2) UC Davis-KARE, U.S.A.; (3) UC Davis, U.S.A.; (4) UC Davis-KARE, U.S.A.
Interactions between pathogenic and nonpathogenic fungal species in the tree canopy are complex and can determine if disease will manifest in the plant and in other organisms such as honey bees. Seasonal dynamics of fungi were studied in almond and pistachio in California. In an almond orchard, experimental release of the atoxigenic biopesticide *Aspergillus flavus* AF36 to displace toxigenic strains has been conducted for five years. Because *A. flavus* is a pathogen of honey bees, our emphasis was on the presence of *A. flavus* in the flowers and the honey bees that attend them. Fungal species most commonly recovered from developing pistachio fruits were: *Cladosporium herbarum*, *Alternaria alternata*, *Penicillium* spp., *Botrytis cinerea*, *Aspergillus niger*, *A. flavus*, and yeasts. The main seasonal changes were detected in *P. expansum* and *A. niger*, which markedly increased from April to September. Conversely, the *B. cinerea* population significantly decreased during this period. In the almond, the dominant genera were: *Cladosporium*, *Aureobasidium*, *Alternaria*, *Aspergillus*, and *Fusarium*. Surprisingly, the diversity index was greater in the summer for both crops. The incidence of almond flowers with *A. flavus* ranged from 0 to 4.5%, depending on the year of study, and the incidence of honey bees carrying *A. flavus* ranged from 6.5 to 10%. This study helps the understanding of seasonal dynamics of pathogenic and nonpathogenic fungi and the impact of AF36 to pollinator honey bees.

## Leaf applications of *Clonostachys rosea* reduce the populations of *Botrytis cinerea* and changes the metabolic profiles of strawberry plants

L. Maffia (1), A. Borges (2), W. Araujo (1)

(1) Universidade Federal de Vicoso, Brazil; (2) Universidade Federal de Vicoso, Brazil

*Clonostachys rosea* is an efficient antagonist of *Botrytis cinerea*. In a research program aiming at the biocontrol of gray mold of strawberry, we studied the effect of *C. rosea* application on both antagonist and *B. cinerea* leaf populations, as well as on plant metabolism at a commercial strawberry field production. The antagonist was applied at 7, 14, or 28-day intervals. *B. cinerea* was naturally inoculated, and the colonization of leaves by both fungi was assessed. The antagonist reduced *B. cinerea* colonization; most significant effects were found with week applications. The reduction in *B. cinerea* colonization was negatively correlated with the increase in *C. rosea*'s at all intervals. The metabolic profiles of treated strawberry plants were also most changed with week applications, and there was a significant increase of compounds related to the Krebs cycle, growth promotion and the activation of plant defense system. In conclusion, with week applications of *C. rosea*, there was a significant reduction on the population of *B. cinerea* in symptomless tissues and a change in the metabolic profile of strawberry plants. Sponsored by CNPq and FAPEMIG.

## Reduction of soybean sudden death syndrome (*Fusarium virguliforme*) by seed treatment with *Bradyrhizobium japonicum*

T. HUYNH (1), T. Huynh (1), X. Yang (1), S. Navi (1), X. Li (1)

(1) Iowa State University, U.S.A.

Soybean sudden death syndrome (SDS), caused by *Fusarium virguliforme*, is a major soil-borne disease in soybean production in America. In North America, epidemics of the disease occurred in years when soybeans were planted early in growing seasons. Previous study by Scherm and Yang (1996) showed that *F. virguliforme* infected soybean in cool soil temperatures. Because infection of symbiont *Bradyrhizobium japonicum* requires minimum temperature of 17.5°C, higher than the minimum for *F. virguliforme* infection, we hypothesize that infections of *F. virguliforme* prior to establishment of *B. japonicum* in soybean affects SDS occurrence in early planting. From 2013 to 2015, experiments were conducted in a greenhouse and fields to determine the effects of seed treatments with *B. japonicum* on the occurrence of soybean sudden death syndrome. Results of greenhouse and field studies showed that seed treatments with *B. japonicum* inoculated reduced SDS foliar symptom expression by 50% and 20% in greenhouse and field, respectively. Increase in soybean nodulation and increase in yield in field experiment were also observed. Our results suggest that seed treatment with *B. japonicum* can reduce SDS foliar symptoms. The mechanisms of the suppressive effects on *F. virguliforme* by *B. japonicum* are under investigation to improve the potential of using this measure for disease control.

## The use of fungal biological control agents to manage SDS, charcoal rot, and damping off in soybean

A. WARNER (1), E. Arnao (2), A. Fakhoury (2)

(1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A.

Sudden death syndrome (*Fusarium virguliforme*), damping off (*Rhizoctonia solani*), and charcoal rot (*Macrophomina phaseolina*) are three important and detrimental diseases of soybean in soybean growing areas. There is a need for more options to control these pathogens; many of the available management options are costly or limited in their effectiveness. Additionally, many of the current control practices may not be sustainable given their unfavorable impact on the environment. The use of biological control agents (BCAs) provides a more natural alternative for controlling various plant diseases. We have evaluated the effectiveness of multiple BCAs against *F. virguliforme*, *R. solani*, and *M. phaseolina*. We have also investigated the mechanisms the BCAs use to control these pathogens. This was determined through the use of microscopy and the monitoring of the expression of a panel of eight mycoparasitism-induced genes.

## Biological control of soilborne diseases in organic potato production using hypovirulent strains of *Rhizoctonia solani*

R. LARKIN (1)

(1) USDA-ARS, U.S.A.

Soilborne diseases are persistent problems in potato production and alternative management practices are needed, particularly in organic production, where control options are limited. Selected biocontrol organisms, including two naturally-occurring hypovirulent strains of *Rhizoctonia solani* (Rhs1a1 and Bs69) and a commercially available *Bacillus subtilis* (GB03), were evaluated, both individually and in combination, for control of soilborne diseases of potato under organic production practices over three field seasons in Maine. Overall, multiple biocontrol treatments resulted in modest but significant reductions in all three soilborne diseases observed (black scurf, common scab, and silver scurf), reducing incidence and severity of each by 10-32%. All biocontrol treatments reduced black scurf (by 15-32%), whereas only specific treatments reduced common scab and silver scurf (by 10-25%). Combinations including both a hypovirulent strain and GB03 tended to be the most effective treatments, although generally not significantly better than individual strains. Tuber yield varied somewhat by year, but Rhs1a1 and GB03 treatments, as well as combinations, increased yield by 10 to 24% overall. These results indicate that hypovirulent Rhizoctonia, along with other biocontrol organisms, may be useful for reducing certain soilborne diseases and enhancing yield in organic potato production.

## Evaluation of Management Practices on the Efficacy of *Pasteuria nishizawae* As a Biocontrol Agent of Soybean Cyst Nematode

M. LUND (1), S. Conley (1), J. Ané (1)

(1) University of Wisconsin-Madison, U.S.A.

Soybean Cyst Nematode (*Heterodera glycines*) is the most economically significant pathogen on soybean (*Glycine max*), causing over $1.5 billion dollars in damages annually. *Pasteuria nishizawae*, an endospore-forming soil bacterium, is an obligate parasite of soybean cyst nematodes (SCN). *P. nishizawae* has the potential to provide season-long control of SCN in the form of a seed treatment called Clariva® Complete. In this study, we evaluated the efficacy of the active ingredient, *P. nishizawae*, for the management of SCN on soybean. The field design was a split-plot, randomized complete block design with sub-sampling for the 2014 and 2015 growing seasons. Each split-plot was randomly assigned a row spacing of 30-inch or 15-inch spacing. Each subplot within the split-plot was randomly assigned one of three seeding rates (75K seeds/acre, 150K seeds/acre and 225K seeds/acre) and one of two seed treatments (Clariva® Complete and CruiserMaxx®/Vibrance®). Results showed no significant interaction between seed treatment and yield, but did show significant interactions between both seeding rate and row spacing with yield. No significant interaction was observed between the
SCN count and the yield. Because the soybean variety used had PI88788 resistance, the Hg-type of the SCN population may not have been able to develop on soybeans with this source of resistance. This may have masked the effects of the seed treatment and prevented accurate evaluation of its efficacy.

## Effect of biopesticides on *Phytophthora* Root Rot Disease of Oakleaf Hydrangea

M. KABIR (1), P. Liyanapathiranage (1), T. Simmons (1), F. Baysal-Gurel (1)

(1) Tennessee State University, U.S.A.

*Phytophthora* root rot, caused by *Phytophthora nicotianae*, has been reported to infect almost all hydrangeas especially container-grown oakleaf hydrangea (*Hydrangea quercifolia*). The pathogen can be introduced into the nursery via *Phytophthora*-contaminated container stock or cuttings. Experiments were performed in a greenhouse to determine the efficacy of biopesticides and fungicide against *Phytophthora* root rot in oak leaf hydrangea vs. Queen of Hearts and Munchkin. Treatments applied were the biopesticides RootShield WP (*Trichoderma harzianum* Rifai strain KRL-AG2) and RootShield Plus (*T. harzianum* Rifai strain T-22 and *T. virens* strain G-41) and fungicide Subdue Maxx (Mefenoxam). All the treatments reduced root rot disease severity compared to the inoculated, non-treated controls in both cultivars. There were no significant differences between the treatments. Root rot disease severity was significantly higher in the cultivar Queen of Hearts than cultivar Munchkin. There was no significant effect of treatment and cultivar on root biomass. This study shows that biopesticides, RootShield WP and RootShield Plus, should be considered to reduce the risk of *Phytophthora* root rot on container-grown oakleaf hydrangeas in the nursery.

## Putative biocontrol mechanisms of rice bacterial blight by *Bacillus subtilis* TKS1-1

T. HUANG (1), Y. Chen (1), Y. Jan (1), Y. Yeh (1), D. Tzeng (1)

(1) Department of Plant Pathology, National Chung Hsing University, Taiwan

Bacterial blight of rice caused by *Xanthomonas oryzae* pv. *oryzae* is one of the devastating diseases of rice plant. The main objectives of the research are to investigate the potential of using *Bacillus subtilis* TKS1-1 as a control agent of rice bacterial blight and the possible mechanisms involved in disease control. *B. subtilis* TKS1-1 showed antagonistic activity against *X. oryzae* WF6. Application of *B. subtilis* TKS1-1 at the time of or prior to inoculation with *X. oryzae* WF6 was found to suppress rice bacterial blight. Additionally, the lesion length of leaf blight was less by drenching application with *B. subtilis* TKS1-1 compared to the water control. Spray-application of *B. subtilis* TKS1-1 was shown to increase the expression of several plant defense-related genes including *PR1a*, *PAL*, *NPR1*, and *WRKY45* at 16 hours post application compared to the water control. Furthermore, drenching application of strain TKS1-1 7 days prior to inoculation of the pathogen was found to increase the activity of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase at 48 hours post-inoculation in comparison with the water treatment. Application of *B. subtilis* TKS1-1 on rice cultivar TNG67 showed significant promotion of plant growth. The findings suggested that *B. subtilis* TKS1-1 exhibited efficacy in suppression of rice bacterial blight and in growth promotion. The biocontrol potential is partly attributed to the induction of plant defense response by *B. subtilis* TKS1-1.

## Amino acids influence antibiotic production by *Pantoea vagans* strain C9-1

J. KLEIN (1), V. Stockwell (2), J. Loper (2)

(1) Oregon State University, U.S.A.; (2) USDA-ARS, Horticultural Crops Research Unit, U.S.A.

*Pantoea vagans* strain C9-1 is an effective biological control agent for fire blight caused by *Erwinia amylovora*. C9-1 is thought to suppress *E. amylovora* on stigmas via resource competition and production of the antibiotics pantocin A (formerly herbicolin O) and herbicolin I. We used a green fluorescent protein reporter construct in C9-1 to monitor the regulation of the promoter of the pantocin A operon (*paaPABC*) on solidified media and in broth culture with a Tecan Infinite M200PRO. In assays on solidified media, inhibition of *E. amylovora* by C9-1 and expression of the *gfp* reporter for pantocin A operon was detected on Neidhardt's modified medium, but not on Luria Bertani (LB) medium. Addition of yeast extract or tryptone (components of LB) to Neidhardt's broth medium suppressed the expression of the pantocin A promoter. We tested the effect of various amino acids (100 mM) added to Neidhardt's broth medium on expression of the *paaPABC* promoter in C9-1. We found that arginine, histidine, or tryptophan reduced expression of the pantocin A promoter compared to expression in non-amended Neidhardt's broth medium, whereas asparagine, aspartic acid, or proline upregulated expression. Asparagine and proline are two major amino acids reported on apple and pear flower stigmas. These results indicate that the chemical environment of stigmas likely would support expression of the *paaPABC* operon and pantocin A biosynthesis by the biological control agent C9-1.

## Microscopic analysis of parasitism mode of action by bacterial biological control agents on *Cornus florida* powdery mildew

M. MMBAGA (1)

(1) Tennessee State University, U.S.A.

Three bacterial isolates, *Baccillus* (sp., *Stenotrophomonassp. Serratia* sp. were previously identified as naturally occurring biological control agents (BCA) for powdery mildew in *Cornus florida*. The interaction between these BCA's and powdery mildew fungus was studied using scanning electron microscopy (SEM) and detached leaf technique. Four replicates of similar leaves were infected with powdery mildew, sprayed with BCA's, incubated for 3h, 6h, 15h, 48h and 5 days, then fixed overnight using formalin acetic acid alcohol and stored at 4°C untill SEM imaging was done. The SEM micrographs showed the three BCA's colonizing powdery mildew spores and hyphae. The BCA's displayed the ability to enter and lyse powdery mildew spores and hyphae; the *Baccillus* sp. lysed and destroyed mycelial structures completely within 48h; others took longer. The BCA's multiplied on the leaf surface and inside powdery mildew spores and hyphae. The three BCA's did not interfere with other microbes on the leaf surface; this displayed potential specificity to powdery mildew and potential preservation of other naturally occuring microflora. The collapsed conidia and lysed hyphae suggested antifungal compounds that caused structural damage may be involved. These observations confirm that the three naturally occuring BCA's may provide a buffering effect against powdery mildew and a resource for powdery mildew management and reducing conventional fungicides usage.

## Investigation of antifungal and herbicide volatile terpenes in *Streptomyces* spp.

G. CHO (1), H. Kim (1), J. Kim (2), J. Kim (1), Y. Kwak (2)

(1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea

*Streptomyces* spp. produce various secondary metabolites for interaction with environment. Some of these secondary metabolites have been reported as antibiotics or volatile compounds. In addition, *Streptomyces* spp. have been highly expectation for discovering new secondary metabolites. We observed that mycelia of various fungi were inhibited growth when these fungi were exposed to headspace of *Streptomyces* spp. Furthermore, germination and growth of *Arabidopsis thaliana* Col-2 were inhibited significantly by these headspaces. In these headspaces, three volatile terpenes that geosmin, caryolan-1-ol and unknown terpene were discovered in common. To confirm the bioactivity, we extracted mixtures from headspace of *Streptomyces griseus* S4-7. Mixture included geosmin didn't inhibit mycelia growth of *Botrytis cinera*. A mixture included caryolan-1-ol and unknown terpene inhibited these mycelia. Furthermore, synthetic caryolan-1-ol caused dose-dependent inhibition of these mycelia and *Saccharomyces cerevisiae* growth. In addition, we found three terpene cyclase gene in *S. griseus* S4-7 genome. Geosmin and caryolan-1-ol synthases already had been reported but the other terpene synthase was unknown. Our present results show that caryolan-1-ol was discovered new bioactivity function.
# Characterization and Expression of the Potent Antimicrobial Peptide LsGRP1C Originated from *Lilium*

C. CHEN (1), C. Lin (1), Y. Pan (2), M. Chang (2), F. Liu (2)

(1) National Taiwan University, Taiwan; (2) National Taiwan University, Taiwan

LsGRP1 is a plant defense-related gene increasingly expressed in lily leaves exhibiting salicylic acid-induced systemic resistance against pathogenic fungus *Botrytis elliptica*. *In vitro* antimicrobial assay applied with synthetic peptides revealed that the 38-a.a. cysteine-rich C-terminal portion (LsGRP1C) of LsGRP1 inhibited kinds of bacterial and fungal species *via* the mechanisms of alteration of microbial membrane permeability and induction of fungal programmed cell death (PCD) as investigated by SYTOX Green staining and 4',6'-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Then the expression system for recombinant LsGRP1C production was established using an optimized *Escherichia coli* strain and an N-terminal fusion partner of small ubiquitin-like modifier protein (SUMO) Smt3 originated from yeast, conducting a high yield of SUMO-LsGRP1C fusion protein. According to the half inhibitory concentrations for spore germination and PCD occurrence of the assayed fungal species, SUMO-LsGRP1C was proven to exhibit higher antifungal activity and identical fungal PCD-inducibility as compared with synthetic LsGRP1C. Thus, we proposed that LsGRP1C would play a role in antimicrobial function of LsGRP1; moreover, efficient and effective *E. coli* expression strategy would enable the convenient production of antimicrobial LsGRP1C for practical use.

# Functional analyses of the ribosomal and non-ribosomal processed antimicrobial peptides produced by biocontrol bacterium *Bacillus pumilus* PMB102

W. DENG (1), J. Wu (1), J. Huang (1)

(1) National Chung Hsing University, Taiwan

Many *Bacillus* spp. are beneficial bacteria with potential applications in the integrated management of plant diseases, and the efficacy of biocontrol is mainly determined by the production of secondary metabolites, e. g. the ribosomal processed lantipeptides and non-ribosomal processed cyclic lipopeptides (CLPs). In this study, the biocontrol agent *B. pumilus* PMB102 was shown to reduce tomato Fusarium wilt disease in hydroponic seedling assays. Annotation of the draft genome identified one lantipeptide and two CLPs, surfactin and plipastatin (fengycin family), biosynthetic gene clusters. Partially purified proteins from the lantipeptide-containing culture filtrate were resolved by electrophoresis and assayed for antibacterial activity by overlay assays, and one small polypeptide was identified to inhibit the growth of *B. cereus* and *B. mycoides*. The culture filtrates containing CLPs were extracted by n-butanol and fractionated by C18 SPE column. The eluted fractions were resolved by TLC and assayed for antifungal activity by the overlay assays to show that the compounds sharing the same Rf value of fengycin could inhibit the mycelial growth of *Alternaria brassicicola*. The chemical compositions of the abovementioned antimicrobial compounds will be further verified by mass spectrometry. The expression of defense-associated genes will also be monitored to determine if the plant defense responses against fungal pathogens are modulated by the biocontrol bacterium PMB102.

# Study on Colonization of Strain ZY-9-13 labeled with Green Fluorescent Protein(GFP) Gene within Roots of tobacco and its Biocontrol Effect

J. XI (1), J. Song (1), M. Mo (2), C. Xue (1), W. Mou (1), Q. Yin (1)

(1) Zhengzhou Tobacco Research Institute of CNTC, China; (2) Yunnan University, China

Strain ZY-9-13 was isolated from fresh leaves of tobacco, which had considerably antagonism to Phytophthora parasiticavar. nicotianae. The stability of the control effect mainly depends on whether antagonistic microbes could effectively colonize in plant. The purpose of the study was to explore the colonization of stain ZY-9-13 in roots of tobacco and control effect to tobacco black shank. The pattern of colonization of the tobacco was observed with fluorescent confocal microscopy and control effect was calculated after the tobacco seedling being inoculated the GFP tagged stain on tobacco black shank. The results showed that there was no obvious difference between original strain ZY-9-13 and tagged strain in antibacterial activities testing. Fluorescence microscope observation indicated that the strain ZY-9-13 propagated in the zone of root cap, elongation, maturation, and the junctions of primary roots and lateral roots. The quantities of the stain in different parts were root cap> elongation > maturation > the junctions. The pot experiment data showed that application of ZY-9-13 significantly reduced the disease incidence of black shank on tobacco. Five doses of the strain ZY-9-13 (1 × 10⁶ cfu·g⁻¹) × 107 cfu·g⁻¹) × 108 cfu·g⁻¹) × 109 cfu·g⁻¹) × 1010 cfu·g⁻¹) showed different control effect on tobacco black shank, which was 42.43%, 54.70%, 68.80%, 73.03% and 77.60% respectively. Results provided scientific basis for the strain ZY-9-13 on controlling tobacco black shank.

# Developing a quantitative polymerase chain reaction protocol to quantitate root colonization by *Bacillus amyloliquefaciens* and *Bacillus firmus*

H. MENDIS (1), L. De La Fuente (1), P. Schwientek (2), R. Salamzade (2), V. Thomas (2), J. Kloepfer (3)

(1) Auburn University, U.S.A.; (2) Bayer CropScience LP, U.S.A.; (3) Auburn University, U.S.A.

*Bacillus amyloliquefaciens* QST713 and *B. firmus* I-1582 are 2 key bacterial strains which are active ingredients in commercially-available biological products that colonize plant roots promoting plant growth and offering protection against pathogens. This study was carried out to develop a qPCR protocol to understand the dynamics of root colonization by these strains. Primers and TaqMan probes were designed based on genome comparisons of the 2 strains with other publicly available bacterial genomes. The specificity of the primers was tested in vitro and in growth chambers focusing on corn rhizosphere. An optimized qPCR protocol was developed to quantify the number of bacteria in batch cultures and in root samples using standard curves. Corn seeds were treated with 10³, 10⁵ and 10⁷ spores of the 2 strains separately and planted in sterile sand. Whole root samples were taken at 2 and 3 weeks after germination and bacterial populations were assessed. QST713 and I-1582 primers did not amplify DNA extracted from untreated corn grown soil confirming that the primers are specific. The results also show that as low as 3.5 × 10³ bacteria cells associated with 1g of corn roots can be detected using the current qPCR protocol. Furthermore, the number of bacteria cells associated with corn roots was quantified up to 3 weeks after germination. Our results show that the developed qPCR protocol can be used to understand dynamics of root colonization by plant growth promoting bacteria.

# Effects of seed coating “inert ingredients” in the pathogenicity of *Fusarium graminearum* in soybean

K. Navarro (1)

(1) The Ohio State University, U.S.A.

*Fusarium graminearum*, known to infect wheat, barley, corn, and other cereal crops has also been reported to cause seedling rot, root rot and pre- post emergence, damping off of soybean. Application of fungicides and other bioactive molecules are often combined with an “inert” ingredient such as sugar polymer dextrans as a seed coat to control seedling pathogens. Our study aims to investigate the effects of maltodextrin seed coating on the virulence of *F. graminearum* in soybean. Five soybean cultivars with different levels of resistance were coated with maltodextrin and inoculated with a microconidia suspension in a roll—towel assay. Our study demonstrates that maltodextrin coated soybean seeds had increase susceptibility to *F. graminearum*. Treated seedlings grew less vigorously in soil than non-treated seedlings. To address if fungal growth rates increased in the presence of maltodextrin, water agar plates amended with maltodextrin were prepared. Fungi grew faster in the presence of maltodextrin and exhibited a change in color as compared to non-amended water agar plates. Further experiments testing to address whether maltodextrin and other related sugar polymers used in seed coatings can affect fungal growth and toxin production are underway. Our study provides information about the potential for unintended adverse effects of specific “inert” ingredient in the formulation of seed coatings.

# The effects of seed coating “inert ingredients” in the virulence of *Fusarium graminearum* in soybean

K. NAVARRO (1)

(1) The Ohio State University, U.S.A.
*Fusarium graminearum*, known to infect wheat, barley, corn, and other cereal crops has also been reported to cause seedling rot, root rot and pre-post emergence, damping off of soybean. Application of fungicides and other bioactive molecules are often combined with an "inert" ingredient such as sugar polymer dextrans as a seed coat to control seedling pathogens. Our study aims to investigate the effects of maltodextrin seed coating on the virulence of *F. graminearum* in soybean. Five soybean cultivars with different levels of resistance were coated with maltodextrin and inoculated with a microconidia suspension in a roll-towel assay. Our study demonstrates that maltodextrin coated soybean seeds had increase susceptibility to *F. graminearum*. Treated seedlings grew less vigorously in soil than non-treated seedlings. To address if fungal growth rates increased in the presence of maltodextrin, water agar plates amended with maltodextrin were prepared. Fungi grew faster in the presence of maltodextrin and exhibited a change in color as compared to non-amended water agar plates. Further experiments testing to address whether maltodextrin and other related sugar polymers used in seed coatings can affect fungal growth and toxin production are underway. Our study provides information about the potential for unintended adverse effects of specific "inert" ingredient in the formulation of seed coatings.

## Predicting the geographic establishment of introduced mycoparasites

S. COHEN (1)

(1) Center for Regulatory Research, LLC, U.S.A.

The mycoparasites, *Coniothyrium minitans* and *Trichoderma viride*, of the plant pathogens, *Sclerotinia sclerotiorum* and *Sclerotinia minor*, offer a model system for the development of an approach for predicting their geographic distribution based on the physiological responses to the environmental variables of temperature and soil moisture. A biome analysis was performed to validate presence-absence data followed by a species response to environmental parameters using CLIMEX, a climate modeling and mapping software. Biome analysis identified the % of biome types capable of supporting *Coniothyrium minitans* (21.4%), *Trichoderma viride* (14.3%), *Sclerotinia minor* (28.6%) and *Sclerotinia sclerotiorum* (21.4%). Geographic distribution of *Sclerotinia* species was based on carpogenic germination of sclerotia in response to temperature and soil moisture parameters. Mycoparasite geographic distribution was determined by conidia germination with respect to the parameters. CLIMEX model analysis reports the US locations that are very favorable for establishment for *Sclerotinia sclerotiorum* (47%), *Sclerotinia minor* (27%), *Coniothyrium minitans* (53%) and *Trichoderma viride* (20%). Areas deemed very favorable for establishment of *Coniothyrium minitans* (53%) were larger than for *Trichoderma viride* (20%). Only 3% of the areas were classified as unsuitable for *Coniothyrium minitans* as compared to 17% for *Trichoderma viride*.

## Differential Colonization of Tomato Cultivars by the Biocontrol Agent *Beauveria bassiana*

S. YERUKALA (1), M. Dee (1), B. Owleyn (1)

(1) University of Tennessee, U.S.A.

Entomopathogenic *Beauveria bassiana* (Bb1198) is endophytic in tomato. Biocontrol of disease with Bb1198 is correlated with endophytic colonization. The aim of this study was to determine if colonization by Bb1198 is affected by tomato cultivar. Colonization of 'Rutgers' and 'Mountain Spring' by Bb1198 was tested in a gnotobiotic assay. Seed were treated with Bb1198 in 1% methylcellulose at 1.6 × 10⁶ viable CFU/seed. Control seeds received methylcellulose. Replicated tests were factorials, in a CRD. Seed germination, BB1198 colonization, and plant growth were determined 17-21 days after treatment. Bb1198 was recovered from sections of root, stem, and leaf plated on semi-selective medium. Data were analyzed with mixed models ANOVA, and F-LSD (P<0.05). Germination was higher for 'Mountain Spring' seed treated with Bb1198 than control seed (94 vs 65%), germination of 'Rutgers' treated and control seed did not differ. Bb1198 was not recovered from plants from control seed. For treated seed, colonization of 'Rutgers' was greater than 'Mountain Spring' (64 vs 29%). For both cultivars from treated seed, colonization of leaf (50%) and stem sections (67%) was greater than roots (23%). Leaf number was greatest in 'Rutgers' from seed treated with Bb1198, followed by control plants for both cultivars, and least in 'Mountain Spring' treated with Bb1198. Understanding factors that affect colonization, i.e., cultivar, could increase consistency of biocontrol by *B. bassiana*.

## Effect of seedborne *Xanthomonas translucens* on wheat seed germination

C. PEREZ (1), E. Juanicotena (2), O. Bentancur (2), C. Palladino (2)

(1) EEMAC, Universidad de la Republica, Uruguay; (2) EEMAC, Universidad de la Republica, Uruguay

Bacterial leaf streak (BLS) has become an important disease on wheat production in Uruguay. However, very little is known about the epidemiology of this diseases. Seedborne pathogens may play an important role as primary source of inoculum but can reduce seed germination as well. The aim of this study was to investigate the effect of *X. translucens* on wheat seed germination. Seeds were collected from nine different commercial plots harvested in 2013, including different cultivars. Seeds lots were taken to the laboratory to test four seed treatments: i) check control (without treatment), ii) disinfectant (didecyldimethyl-ammonium chloride), iii) fungicide (iprodione + TMTD + carbendazim), vi) disinfectant + fungicide. Bacterial density was estimated by seed washing and dilution plating technique on Wilbrink's agar. Seed germination was performed on paper towel. Significant differences on bacterial density were found among seed lots. Seed treatment significantly reduced the number of cfu.g⁻¹ of seed, being disinfectant the most effective reducing bacterial population. However only the fungicide showed a significant effect on seed germination, indicating that germination was not affected by the bacterial population present at the seed surface. Based on this preliminary result, seedborne *X. translucens* might play an important role as source of inoculum but did not affect germination.

## Characterization and Application of Oil-In-Water Nanoemulsion for Controlling Citrus Huanglongbing

M. ZHANG (1), C. Yang (2), C. Powell (1), Y. Duan (3), R. Shatters (3)

(1) IRREC-IFAS, University of Florida, U.S.A.; (2) Guangxi University, China; (3) USHRL-USDA-ARS, U.S.A.

*Candidatus* Liberibacter asiaticus (Las), which can cause destructive citrus huanglongbing (HLB), resides in citrus phloem. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, oil-in-water (O/W) nanoemulsion was optimized to prepare the antimicrobial nano-formulation. The nanoemulsion was produced using a spontaneous emulsification method for the efficient delivery of antimicrobials into citrus phloem by bark application. The nanoemulsion prepared from cremophor® EL (viscous oil), acetone (water miscibility organic solvent), and Span 80/Tween 80 (surfactant) exhibited a small droplet size (17.33±0.52 nm). It also had an improved absorption rate, including 2-day peak concentration ($t_{max}$), 71.86±35.38 ng/g maximum concentration ($C_{max}$), and 267.25%±44.1% relative bioavailability (RBA) of ampicillin in HLB-affected citrus compared to the Amp solution alone ($t_{max}$ = 6 days, $C_{max}$ = 56.44±32.59 ng/g and RBA = 100%). The same nanoemulsion was used to deliver five antimicrobials to control citrus HLB by bark application. We found that droplet size of antimicrobial formulation prepared in nanoemulsion was significantly reduced. And the nanoemulsion also enhanced the efficacy against Las of validoxylamien A, combination of actidone and validoxylamine A, and sulfadimoethoxine sodium against Las. Therefore, this study provides an efficient self-nanoemulsifying delivery system for controlling citrus HLB.

## Effects of Penicillin Injection for Citrus HLB Control on Culturable Bacteria in Petioles and Rhizosphere, and Penicillin Resistance

K. SHIN (1), M. Ascunce (1), H. Narouei-Khandan (1), X. Sun (2), D. Jones (2), E. Goss (1), A. van Bruggen (1)

(1) University of Florida, U.S.A.; (2) Division of Plant Industry, U.S.A.

Trunk injection with penicillin has been tested to control citrus huanglongbing (HLB), but environmental safety must be assured before approval of penicillin injection in groves. We investigated potential effects of penicillin injection on rhizospheric and endophytic bacterial populations using
grapefruit trees in field and greenhouse experiments. Trees were injected with penicillin G, using application rates of 0, 1000, or 6000 µg/ml. Bacteria were isolated on a low carbon medium from roots plus rhizosphere and surface-sterilized petioles at various times after penicillin injection. Selected isolates were tested for penicillin resistance (20 µg/ml) and glyphosate resistance (7000 µg/ml), because glyphosate is widely used and cross-resistance against antibiotics had been documented. Bacterial populations in petioles and root-rhizospheres initially increased after penicillin injections, possibly due to release of nutrients from dead bacteria, and then returned to control levels after one week. Penicillin resistance was common in penicillin-injected and control trees (30 to 94%). A few isolates (0 to 2%) were only resistant to glyphosate, while more isolates (29 to 30%) were only resistant to penicillin. Most isolates (54 to 71%) were resistant to both glyphosate and penicillin. Considering the widespread penicillin resistance in control soil, potential side effects of penicillin injection in citrus are small.

## Evaluation of a novel antimicrobial compound to control growth and biofilm formation in vitro of citrus bacterial pathogens

H. MENDIS (1), E. Naranjo (1), S. Santra (2), M. Young (2), E. Johnson (3), P. Rajasekaran (2), L. De La Fuente (1)
(1) Auburn University, Auburn, AL, U.S.A.; (2) University of Central Florida, Orlando, FL, U.S.A.; (3) Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.

Zinkicide™ is a novel plant nutrient-based antimicrobial formulation developed for treating citrus bacterial pathogens including *Candidatus Liberibacter asiaticus* (Las) and *Xanthomonas citri* subsp. *citri* (Xac). As per the design, Zinkicide™ is expected to enter phloem tissues of citrus plants when sprayed onto leaves for control of phloem-limited pathogens such as Las. This study investigated the effects of Zinkicide™ in vitro on growth and biofilm formation of Las surrogate *Liberibacter crescens* and Xac in batch cultures and under flow conditions using microfluidic chambers. Xac was grown in 96-well plates in Nutrient Broth and treated with serial dilutions of Zinkicide™ to test inhibition of growth and biofilm formation. Xac was also introduced to microfluidic chambers under constant media flow, and inhibitory action of Zinkicide™ on biofilm formation was recorded using time-lapse video imaging microscopy. The Zinkicide™ formulation had better antimicrobial properties compared to similar formulations with the same active ingredients, and the minimum inhibitory concentration of Zinkicide™ for Xac was calculated at 10ppm. Time-lapse video imaging showed that untreated Xac grown in microfluidic chambers formed biofilm whereas Xac treated with Zinkicide™ did not form any biofilm. In vitro results confirmed that Zinkicide™ is effective against citrus bacterial pathogens and inhibits bacterial growth and biofilm formation under flow conditions.

## Progress towards a novel chemical control for common scab disease of potato

R. TEGG (1), H. Thompson (2), C. Wilson (1)
(1) Tasmanian Institute of Agriculture, University of Tasmania, Australia; (2) Department of Agriculture, Australia

Common scab is a globally important potato disease caused by pathogenic *Streptomyces* spp. with no one effective control strategy. Essential for disease induction is the production by the pathogen of the toxin, thaxtomin A. This study sought to develop disease control options targeting the interaction of thaxtomin A with the host, using novel chemical applications. Trials identified two compounds, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,5-dibromobenzoic acid (2,5-DBB) that when applied to the foliage of newly emerged plants prior to tuber initiation, could significantly reduce common scab tuber disease incidence and severity by over 85%, compared to the untreated control. Seed tuber treatments with 2,4-D prior to planting were also effective at reducing disease although the level of control was not as great as from foliar treatments. Disease resistance was correlated with decreased sensitivity of the tuber material from treated plants to thaxtomin A. High application rates (>200mg/L) of 2,4-D were associated with negative traits including twisted foliage, tuber deformation and yield loss. Further trials demonstrated that greatly reduced 2,4-D application rates down to 1.7-25mg/L retained effective disease control but avoided detrimental phytotoxicity effects. Field trials within Australia, China and USA testing foliar and seed treatments are on-going, with initial results indicating useful field efficacy of these chemical treatments for disease control.

## Antibacterial potential of Magnesium oxide nanomaterial against *Xanthomonas perforans* causing bacterial spot of tomato

Y. LIAO (1), A. Strayer (1), J. White (2), A. Mukherjee (2), W. Elmer (3), L. Ritchie (4), D. Clark (4), J. Freeman (4), J. Jones (1), M. Paret (4)
(1) Department of Plant Pathology, University of Florida, Gainesville, FL, U.S.A.; (2) Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (3) Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (4) North Florida Research and Education Center, University of Florida, Quincy, FL, U.S.A.

Bacterial spot caused by *Xanthomonas perforans* can cause high disease severity on tomatoes in Florida. Due to the presence of copper (Cu)-tolerant *X. perforans* strains, copper-bactericides are of limited use; thus, there is a critical need for finding effective alternatives. In this study the antibacterial activity of magnesium oxide (MgO) and other metal oxide nanomaterials were evaluated against Cu-tolerant and sensitive *X. perforans* strains. In vitro assays demonstrated that MgO had high antibacterial ability compared to the equivalent concentration of a Cu-based bactericide against both strains. A greenhouse experiment demonstrated that disease severity was significantly reduced by MgO at 200 ppm compared to copper-mancozeb, the grower standard and the untreated control (p = 0.05). A field experiment demonstrated that the same treatment significantly reduced disease severity compared to the untreated control (p = 0.05). There was no significant difference in the yield between the treatments. Inductively Coupled Plasma Mass Spectrometry analysis of the fruits showed that MgO treatments did not lead to significant accumulation of Mg, Cu, Ca, K, Mn, P and S compared to the untreated. However, copper-mancozeb treatment led to significant accumulation of Cu and P compared to the untreated control (p = 0.05). This study shows the antibacterial potential of MgO nanomaterials against *X. perforans* and its potential use against bacterial spot of tomato.

## Adepidyn™: A new fungicide active ingredient for control of foliar diseases

K. BUXTON (1), T. Harp (1), A. Tally (1), H. Mclean (1)
(1) Syngenta Crop Protection, U.S.A.

Adepidyn™ fungicide is a new active ingredient in the carboxamide chemical class (FRAC group 7) from Syngenta that provides excellent, long lasting control of powdery mildews, leaf spots, and other fungal pathogens on vegetables as well as row crops. The first wave crops to be registered include fruiting and leafy vegetables, cereals, peanut, grapes, potatoes, soybeans, and corn. Some of the key strengths of ADEPIDYN™ include early and late leaf spot on peanut, *Alternaria* spp., and powdery mildews. Field trials have shown the unparalleled residual control of ADEPIDYN™ fungicide, at low rates in comparison to commercial standards, providing more than three to four weeks control of *A. solani* on potato, *Cercospora* spp. on peanut, and powdery mildew on cucurbits. The high intrinsic activity and long-lasting duration of control of ADEPIDYN™ fungicide on these diseases, in rotation and in mixtures with other fungicides, will provide growers another tool for effectively managing leaf spot and powdery mildew diseases.

## Adepidyn™: A new fungicide active ingredient for control of Fusarium head blight on wheat

T. HARP (1), K. Anaka (2), A. Tally (1)
(1) Syngenta Crop Protection, U.S.A.; (2) Syngenta Crop Protection, Canada

Adepidyn™ Fungicide is a new active ingredient in the carboxamide chemical class (Frac group 7) from Syngenta that provides broad spectrum disease control across many crops, including cereals. In addition to strong performance on various cereal leaf spots and powdery mildew, Adepidyn™ has also demonstrated efficacy on Fusarium head blight (FHB), a disease that is difficult to control and with limited management solutions. Adepidyn™ represents the only carboxamide that demonstrates good activity for control of FHB, offering growers a new tool for Fusarium control as well as providing an expanded window for application. In field trials, Adepidyn™ applied solo between BBCH 55 and BBCH 65 (Feekes 10.3-10.52) at rates between 150 and 200 gai/ha, or at 125 to 150 gai/ha in mixture with propiconazole, provided superior efficacy to the current triazole solutions. Adepidyn™ provides excellent control of FHB and is a new mode of action for effective management for FHB in cereals.
## Quantification of the mortality of conidia of *Botrytis cinerea* and *Penicillium digitatum* in peracetic acid

J. Smilanick (1), L. Grant (2)

(1) consultant, U.S.A.; (2) Jet Harvest Solutions, U.S.A.

Peracetic acid (PAA) is common sanitizer, but information about its fungal toxicity is sparse. PAA toxicity was assessed to conidia of *Botrytis cinerea* and *Penicillium digitatum*, causes of gray mold and citrus green mold, respectively. They were immersed in PAA, entrapped on a glass fiber filter, rinsed with dH₂O, and placed on PDA to observe germination, then the time (LT₀₅) or concentration (LD₀₅) when 95% mortality occurred were estimated by probit analysis. Two 100 mg/l (= ppm) solutions were used, one from 4.9% PAA + 26.5% H₂O₂ and the other from 15.0% PAA + 22.0% H₂O₂. Mortality was similar in both therefore due to PAA with little or no H₂O₂ contribution. ET₀₅ of *B. cinerea* and *P. digitatum* conidia in 100 mg/l PAA was 5.4 and 2.4 minutes, respectively. ET₀₅ of *B. cinerea* and *P. digitatum* conidia in 500 mg/l PAA was 17.2 and 9.8 seconds, respectively. LD₀₅ of *B. cinerea* and *P. digitatum* conidia during a 24-hour PAA exposure was 24.7 and 3.3 mg/l, respectively. LD₀₅ of *P. digitatum* conidia during 10 or 30-minute PAA exposure was 50 and 41 mg/l, respectively. Although PAA resistance was markedly higher in *B. cinerea* than in *P. digitatum*, label rates of PAA are adequate to control conidia of both pathogens. PAA can be mixed with fungicides, label rates are relatively high (650 mg/l preharvest and 85-100 mg/l postharvest), is USDA Organic Program organic rules, and its disinfection by-products are of minimal concern.

## Effect of fungicide applications on *Monilinia fructicola* population diversity and transposon movement

M. DOWLING (1), H. Boatwright (1), G. Schnabel (1), P. Bryson (1), J. Wilson (1), Z. Fan (1), S. Everhart (3), P. Brannen (4)

(1) Clemson University, U.S.A.; (2) Clemson University, Clemson, SC, U.S.A.; (3) University of Nebraska, Lincoln, U.S.A.; (4) University of Georgia, U.S.A.

In this study we investigated whether previously reported, fungicide-induced mutagenesis observed *in vitro* in *M. fructicola* can also accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied at half label rate to nectarine trees for two consecutive years in weekly intervals for three months between bloom and harvest. Single spore isolates were obtained from blighted blossoms, corresponding cankers, and from fruit to investigate phenotypic and genotypic changes. In both years, isolate populations collected from fungicide-treated fruit and from untreated control fruit were not statistically different in haploid gene diversity (p > 0.45 for 2013), (p > 0.46 for 2014), allele number (p = 0.88 for 2013), (p = 0.41 for 2014), and effective allele number (p > 0.88 for 2013), (p > 0.81 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments had no changes in simple sequence repeat size or evidence for induced transposon translocation. No emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil that would indirectly indicate increased genetic diversity was detected. Our results indicate that fungicide-induced, genetic changes may not occur as readily in field populations as they do in populations exposed continuously to sublethal doses *in vitro*.

## Citrus Canker as a bioassay for systemic bactericidal activity of Zinc nanoparticles

S. COMMERFORD (1), K. Gerberich (1), P. Rajasekaran (2), M. Young (3), S. Das (3), J. Graham (4), S. Santra (3), E. Johnson (1)

(1) Citrus Research Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (2) University of Central Florida, Orlando, FL, U.S.A.; (3) University of Central Florida, U.S.A.; (4) Citrus Research Education Center, University of Florida, U.S.A.

Huanglongbing (HLB) is caused by the phloem-limited bacterium, *Candidatus* Liberibacter asiaticus (Las), transmitted by the Asian citrus psyllid. Since August 2005, when HLB was first discovered in South Florida, citrus production has declined by more than 60%. HLB symptoms include vein yellowing, blotchy mottle, zinc deficiency, tree decline and fruit drop. Like most systemic diseases, HLB is difficult to control because foliar applied materials cannot enter the vascular tissue due to the leaf cuticle and selective permeability of cell membranes. Zinkicide™ is a plant nutrient-based nanoparticle with bactericidal activity that is designed to be translocated systemically. Prior to HLB, citrus canker was the most serious disease threat to Florida citrus. The causal agent, *Xanthomonas citri* subsp. *citri* (*Xcc*), incites leaf and fruit lesions within 2-3 weeks after infection. The goal was to investigate systemic movement of Zinkicide™ via soil drench using canker as a model for bactericidal activity because of the short evaluation time compared to HLB. In replicate greenhouse trials, soil drench and spray applications of Zinkicide™ were applied prior to leaf infiltration with 10⁴ cfu ml⁻¹ of *Xcc*. Two weeks post inoculation lesions per leaf were counted. Zinkicide™ spray and drench applications significantly reduced lesion number. Reduction of lesions by the drench treatment indicates that systemic activity of Zinkicide™ will be useful for further evaluation against HLB.

## Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect against Citrus Huanglongbing via Trunk Injection

J. HU (1), J. Hu (2)

(1) University of Florida/IFAS-CREC, U.S.A.; (2) University of Florida, U.S.A.

Citrus Huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing *Candidatus* Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root systems was achieved 2 days post injection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root systems and leaves of OTC-treated trees were over 95% and 99% (i.e. 1.76 and 2.19 log reduction) between 2 and 28 DPI. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

## Kasugamycin mixtures with copper, mancozeb, or a thiadiazole improve bactericidal inhibition and efficacy of treatments against walnut blight

K. NGUYEN (1), H. Forster (1), L. Wade (2), J. Adaskaveg (3)

(1) University of California Riverside, U.S.A.; (2) Arysta LifeScience, U.S.A.; (3) University of California Riverside, U.S.A.

Resistance to copper is common in *Xanthomonas arboricola* pv. *juglandis* (*Xaj*) populations in California walnut orchards making disease management difficult when applying copper alone. The use of mancozeb in mixtures with copper led to improved disease control. The risk of resistance development to both compounds, however, is high because this has occurred with other *Xanthomonas* spp. This stresses the need for new bactericides. Kasugamycin is an effective antibiotic against several bacterial pathogens (e.g., *Erwinia* and *Pseudomonas* spp.) but strains of *Xaj* often exhibit higher minimal inhibitory concentrations (MICs) than those of other species. Our goal was to improve the activity of kasugamycin by mixing with copper, mancozeb, or a thiadiazole. In *in vitro* spiral gradient endpoint and dilution plate testing with copper-resistant strains of *Xaj*, the average MIC for kasugamycin was 27.5 mg/L. When kasugamycin was mixed with copper, mancozeb, or a thiadiazole, average MIC values were reduced to 11.2, 6.1, or 12.8 mg/L, respectively. In field trials, treatments of kasugamycin mixed with either copper, mancozeb, or a thiadiazole reduced natural disease incidence by 84.2, 81.7, or 87.8%, respectively, whereas kasugamycin alone lowered blight by 50.3%, indicating the importance of using this antibiotic exclusively in mixed treatments for increased performance. Kasugamycin is currently under federal and California regulatory review for full registration on walnut.

## Evaluation of conventional and biological pesticides for managing tomato bacterial spot during transplant production

P. ABRAHAMIAN (1), J. Jones (2), G. Vallad (1)

(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.
Bacterial spot caused by *Xanthomonas perforans*, is a ubiquitous disease of tomato in both field and transplant operations in Florida. The high prevalence of copper-tolerant bacterial strains has compromised the efficacy of traditional copper compounds. Fifteen treatments consisting of various chemical and biological pesticides alone or in programs, including a non-treated control and a copper hydroxide standard, were evaluated for their ability to control bacterial spot on tomato seedlings. Treatments were foliar applied to tomato seedlings grown in 141 cell Speedling trays maintained under conditions similar to a commercial transplant operation. Each treatment was tested at least twice in four, 3-week experiments. Disease severity was evaluated on the third week. Stand-alone applications of Actigard, Cueva, Quintec, Innovotech A, and Innovotech B significantly reduced disease compared to the inoculated controls. Programs that included Actigard + Tanos + Agriphage and Tanos + K-Phite + Cueva also significantly reduced disease compared to the control. The antibiotics, oxytetracycline and streptomycin, and Agriphage were ineffective at reducing disease severity under high disease pressure. Severe phytotoxicity was associated with Quintec at rates greater than 1 fl. oz. The copper formulation in Cueva performed better than the copper hydroxide standard. Overall, Actigard provided significant disease reduction as a stand-alone or in a program with other compounds.

## Nanoparticle encapsulation of seed treatment active ingredients for protection of maize seeds against Fusarium graminearum

L. WASHINGTON (1), A. Mullis (2), D. Mayfield (3), B. Narasimhan (2), G. Munkvold (3)

(1) Department of Plant Pathology Iowa State University, Ames, IA, U.S.A.; (2) Department of Chemical and Biological Engineering Iowa State University, U.S.A.; (3) Department of Plant Pathology Iowa State University, U.S.A.

Fungicide seed treatments have long been a viable and effective strategy for protecting against seedborne and soilborne pathogens. However, losses to seeding diseases are still an important issue and there is a need for improved seed treatment efficacy. Amphiphilic polyanhydride nanoparticles have been used to enhance efficacy for several active ingredients in animal systems and have potential for agricultural use. Nanoparticle encapsulation has the potential to reduce active ingredient dosage and to provide sustained release. In order to assess these potential benefits, we developed a method to load polyanhydride nanoparticles with fungicide active ingredients to protect maize seeds against seedling blight caused by *Fusarium graminearum*. The objectives of this study were to determine the efficacy of a reduced rate of nanoparticle-encapsulated thiabendazole for suppression of seedling blight on hybrid maize seeds. Loaded nanoformulations were applied at varying rates with a planting polymer. These nanoparticle treatments were compared to a non-encapsulated thiabendazole treatment and a control. Seeds were treated and placed in a rolled towel assay for one week, to evaluate the effects on root length, shoot length, plant weight, and disease severity. Preliminary results indicate that the nanoformulated thiabendazole had significantly higher plant weight (p<0.0001) and root length (p<0.0001) than traditional thiabendazole seed treatments and inoculated controls.

## Evaluation of host resistance and chemical control to manage white mold of soybean in Ohio

J. HUZAR NOVAKOWISKI (1), J. Huzar Novakowiski (1), J. Winger (1), P. Paul (1), A. Dorrance (1)

(1) The Ohio State University, U.S.A.

White mold of soybean is caused by the fungus *Sclerotinia sclerotiorum*. In Ohio, localized outbreaks of the disease have occurred in each of the past seven years. Chemical control has had inconsistent results for disease management, especially when applied to cultivars with moderate levels of resistance. Therefore, in order to improve disease management recommendations, the objective of this study was to evaluate the efficacy of chemical control on soybean cultivars with different levels of resistance. The experiment was carried out in two fields in 2014 and five fields in 2015, all with a long history of the disease. Ten chemical treatments (untreated check, four fungicides applied at R1 or R1+R2 growth stages and one herbicide applied at R1), and two to four soybean cultivars were arranged in a split strip randomized block design with eight replications. Two fields had white mold in the untreated plots. There was significant interaction between chemical treatments and cultivars (P<0.05). Disease severity index (DSI) in the trials ranged from 2 to 41 in 2014 and from 14 to 44 in 2015. Applications of Endura fungicide and Phoenix herbicide significantly reduced DSI compared to the untreated control in both years. The cultivars with the highest disease resistance ratings had the lowest DSI and the highest yields. Thus, based on our results, growers should focus on soybean cultivars with the highest resistance ratings to manage white mold of soybean.

## Managing QoI-resistant *Cercospora sojina* in Mississippi soybean and assessing the physiological impacts of foliar fungicide phytotoxicity

J. MANSOUR (1), M. Tomaso-Peterson (2), A. Henn (2), J. Bond (1), T. Irby (2)

(1) Mississippi State University, U.S.A.; (2) Mississippi State University, U.S.A.

Following the widespread detection of quinone outside inhibitor (QoI) resistant frogeye leaf spot (FLS) (*Cercospora sojina* Hara) in Mississippi during 2013 and 2014, effective management of FLS in susceptible cultivars requires the use of fungicide products that contain alternative, non-QoI, modes of action (MOA). One drawback to applying alternative MOAs, such as demethylation inhibitors (DMI), can be phytotoxicity on treated soybean leaves. Field trials were conducted in 2015 to assess fungicide efficacy on QoI-resistant FLS severity and the resulting phytotoxicity on several soybean cultivars with differing FLS reactions. The fungicides chosen have previously been observed to produce moderate to severe phytotoxicity. Six different fungicide products, comprised of single or multiple MOAs, were applied at R2, R3, and R4 along with a non-treated FLS and phytotoxicity were visually assessed pre- and several times post-application. Plant height, number of pods and nodes were recorded to determine physiological impacts. Analyzed data revealed that certain MOAs significantly reduced FLS severity compared to the non-treated. In addition, prothioconazole resulted in significantly greater phytotoxicity than the non-treated, a difference of 29.4%. However, yield was not significantly reduced as a result of fungicide phytotoxicity. In addition, plant heights and number of nodes per plant were not significantly different between fungicide treatments and the non-treated.

## Management of Fusarium Head Blight and DON in Alabama

K. BOWEN (1), N. Sharma (1), N. McMaster (2), J. Jones (3), M. Pegues (3)

(1) Auburn University, U.S.A.; (2) Virginia Tech, U.S.A.; (3) Alabama Agricultural Experiment Station, U.S.A.

Fusarium head blight of wheat (FHB), caused by *Fusarium graminearum*, was severe on winter wheat in southern Alabama in the spring of 2015. A fungicide trial with four replications, planted to 'AGS 2060', allowed evaluations of demethylation inhibitor (DMI) and DMI plus quinone outside inhibitor (QoI) fungicides for managing FHB and the mycotoxin, deoxynivalenol (DON). Fungicides were applied at Feekes stage 10.5 (inflorescence complete). FHB intensity across plots was rated on a scale of 0 to 9 on 27 Apr, where 0=no disease and 9=severe disease. Plots were machine-harvested 5 Jun and kernel samples assayed for DON content. FHB in non-treated plots was rated at 7.2, which was significantly greater than FHB intensity in plots treated with propiconazole (Prop), tebuconazole (Tebu) plus trifloxystrobin (Trif), or Tebu plus prothioconazole (Proth), while Trif+Proth and metaconazole (Metc) plus pyraclostrobin (Pyra) had intermediate FHB levels. DON concentration, however, was > 5 ppm in grain samples from Trif+Proth- and Metc+Pyra-treated plots; only grain from Tebu+Proth-treated plots had lower DON concentrations, at 3.1 ppm, than from non-treated plots (4.6 ppm). Yield and test weight were low due to heavy disease pressure and lodging, and did not differ due to fungicide. These results confirm that a QoI fungicide, even when applied in combination with a DMI, can increase DON concentration under severe FHB pressure in wheat.

## Effect of leaf spot pathogens and fungicide application on Bacterial leaf streak development in wheat under field conditions in South Dakota

S. ALI (1), S. Sehgal (1), K. Glover (1), N. Kaur (1)

(1) South Dakota State University, Brookings, SD, U.S.A.

Bacterial leaf streak (BLS), caused by *Xanthomonas compastris* (Xc) pv. *Undulosa*, is an important disease of wheat in South Dakota. The objective of this study was to see the effect of fungal leaf spot pathogen "Pyrenophora tritici-repentis (Ptr)" (tan spot) and fungicide application on BLS development. To obtain our objectives, two wheat cultivars Briggs (BLS susceptible) and Select (moderately resistant) were planted in a randomized complete block design with three replications at SDSU Volga Agricultural Experimental Station, Volga, SD in 2014. Three plots of each cultivar was
inoculated with; Xc cell suspension alone (T1), Ptr spores suspension and then with Xc (T2), Ptr, Xc and then applied fungicide "Prosaro" (T3) and Ptr
alone (T4), and non-inoculated and untreated (T5). BLS disease data was recorded 12 days post-treatments. BLS disease severity was observed higher in
T2 (50.5%) as compared to T1 (Briggs =23.33%) and T3 (41.83%) in both cultivars though the severity level was higher in Briggs as compared to Select.
Our results indicate that Ptr infection may help in enhancing bacterial penetration. Also, fungicide application stops tan spot development and that may
helps in increasing BLS development.

Control of apple rots caused by *Phacidiopycnis washingtonensis*, *Phacidium lacerum* y *Diplodia seriata* in postharvest in Chile

G. A (1), M. Caceres (1), M. Lolas (1), C. Pacheco (1), T. Daza (1)

(1) Universidad de Talca, Chile

The apple rots caused by fungal species in pre-harvest and postharvest are an important problem for Chilean apple industry. Recently, we detected to *Phacidiopycnis (Pha.) washingtonensis*, *Phacidium (P.) lacerum* and *Diplodia (D.) seriata* associated with apple rot in pre-harvest and postharvest in Maule Region, Chile. The objective this study were determine i) to sensitivity in vitro to fludioxonil (phenyl pyrrole), pyrimethanil (aniline-pyrimidine) tebuconazole (DMI) and thiabendazole (benzimidazole), and ii) to reduce infection with fungicide applied prior to storage on apple cv. Cripps Pink. Apple fruits were protected with different fungicides and methods (fogging and drench), and after 24 h inoculated with each pathogen. All fruit were stored at 0°C for 60 d. The results indicate that the isolates of *Pha. washingtonensis*, *P. lacerum* and *D. seriata* were sensitive to all fungicides, obtaining differents EC₅₀ values. Fludioxonil, pyrimethanil, and thiabendazole by thermo-fogging reduced significantly to infection during cold storage obtaining an efficacy of 75% to 96%. Fludioxonil and thiabendazole applied by drench reached between 84% and 100% of effectivity. Finally, fungicide applied prior to storage by fogging or drench can be an effective control of apple rots during cold storage in Chile.

Reducing initial infections of apple scab and black rot with salicylic acid and Actigard applied as trunk injections or sprays

P. ABBASI (1), G. Braun (2), E. Bevis (2), S. Fillmore (2)

(1) Agriculture and Agri-Food Canada, Canada; (2) Agriculture and agri-Food Canada, Canada

Apple scab [Venturia inaequalis] and black rot [Botryosphaeria obtusa] are two fungal diseases that affect apple leaves early in the growing season and also make fruit unmarketable at harvest. Salicylic acid has been found to induce plant defense mechanisms to reduce initial disease infections. In this study, salicylic acid and Actigard were applied as trunk injections and spray treatments to measure their effects on the development of apple scab and black rot on established apple trees ('Honeycrisp' and 'Cortland') in the field during 2014 and 2015. Treatments were applied at the tight cluster stage of bud development and again near bloom. Incidence of leaf infections was assessed in planta 7, 14, and 21 days after the second treatment and disease incidence on fruit was assessed at harvest. For injection treatments, the branch directly above the injection site was assessed. In 2014, salicylic acid and Actigard applied as trunk injection or spray treatments resulted in lowest black rot and apple scab incidences on leaves and fruit compared to the control treatment. In 2015, salicylic acid and Actigard again showed a lower incidence of apple scab in the early stages of leaf infections only. No reduction in fruit infection was observed in 2015. Although salicylic acid and Actigard treatments showed a reduction in early leaf infections, apple trees must also be protected from secondary infections.

Interruption and reduction of *Erysiphe necator* cleistothecia development utilizing fungicidal oil

L. THIESSEN (1), W. Mahaffee (2)

(1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A.

Ascospores are the primary inoculum source of *Erysiphe necator* in most regions that grapevine is grown and overwinter within cleistothecia that are
formed prior to leaf drop. Preventative fungicides are concluded at véraison (BBCH 83), and *E. necator* has ample time to colonize large portions of the
leaf tissue, mate, and produce cleistothecia. Interruption of *E. necator* sexual reproduction post-véraison using a fungicidal oil may reduce overwintering
inoculum. Chardonnay vines were sprayed once with 1% Organic JMS Stylet Oil (Vero Beach, FL) in 2014 and 2015. Fungicide application treatments
were made when cleistothecia primordia were observed on leaves (BBCH 89), then 7, 14 and 21 days after primordia were observed. Cleistothecia
development was monitored by weekly sampling of 10 leaves, approximately 1.4 m from the ground, per plot beginning at harvest (BBCH 89).
Cleistothecia were enumerated along a transect on the leaf, and treatments were assessed using pairwise comparisons. Stylet oil applications significantly
reduced cleistothecia in both years (P < 0.05) with no effect of application timing in either year (P > 0.4). Despite a reduction in the number of
cleistothecia produced, cleistothecia were present in all plots prior to leaf drop, indicating that there may be limited utility of a late season curative
fungicide application for disease management.

Etiology and management of sour rot in grapes

M. HALL (1), M. Hall (1), G. Loeb (1), W. Wilcox (1)

(1) Cornell University, U.S.A.

Sour rot is a major challenge for affected grape growers and winemakers worldwide, yet basic disease etiology and management techniques are not well understood. Symptoms are characterized by fruit rot accompanied by the smell of acetic acid and presence of *Drosophila* (fruit fly) species. We’ve successfully reproduced these visual and olfactory disease symptoms and the accompanying characteristic production of ethanol and its conversion to acetic acid within affected berries, in the lab. Healthy fruit were wounded, inoculated with *Saccharomyces cerevisiae* and *Acetobacter aceti*, and exposed to *D. melanogaster* adults. Inoculation without exposure to flies significantly promoted ethanol production but not acetic acid generation, whereas concomitant exposure to flies resulted in both. In field trials conducted on cv. ‘Vignoles’ in 2013-15, both insecticide and antimicrobial treatments significantly reduced sour rot development. In 2015, untreated vines averaged 20.5% sour rot severity, and this was reduced by 73-81% on vines treated prophylactically post-veraison with weekly sprays containing a combination of the insecticide zeta-cypermethrin plus the antimicrobial potassium metabisulfite or hydrogen dioxide; severity was reduced by 49% on vines receiving only insecticide sprays. These trials further support the hypothesis that sour rot results from a complex of yeast, bacteria, and *Drosophila*, and that targeting these organisms can provide significant levels of control.

Characterization of *Diaporthe* spp. *Cadophora malorum* and *Neofusicoccum parvum* causing cordon dieback in kiwi plant and their chemical control in Chile

G. DIAZ (1), M. Lolas (1), B. Latorre (2), E. Ferrada (2), J. Zoffoli (2)

(1) Universidad de Talca, Chile; (2) Pontificia Universidad Catolica de Chile, Chile

Kiwifruits is the third largest exporter of fresh kiwifruit (Actinidia deliciosa) worldwide, with over 10,000 ha cultivated. Cordon dieback in kiwi plant has increased during the last decade in Chile. Recently, we have identified several species obtained from cordon dieback in kiwi plants. Therefore, the objectives of this work were i) to characterize by means of molecular and pathogenicity studies and ii) chemical control of Diaporthe ambigua, Diaporthe australafricana, Cadophora malorum and Neofusicoccum parvum. Pure cultures were characterized by phylogenetic analyses using three loci (ITS, β-tubulin and EF1-α). Pathogenicity test were conducted on kiwifruit canes and trunks. In vitro sensitivity and protection of pruning wounds with benomyl, pyraclostrobin and tebuconazole was evaluated. This study reveals that Diaporthe ambigua, Diaporthe australafricana, Cadophora malorum and Neofusicoccum parvum are the major fungal species causing cordon dieback, and that the protection of pruning wounds with chemical fungicide is a good measured to prevent infection in commercial orchards of kiwi plants in Chile.
## Contribution of protectant fungicides applied as mid-season cover sprays to management of peach brown rot at harvest

N. LALANCETTE (1), N. Lalancette (1), L. Blaus (1), J. Gager (1), K. McFarland (2)

(1) Rutgers University, Agricultural Research & Extension Center, U.S.A.; (2) Rutgers University, Agricultural Research & Extension Center, U.S.A.

Protectant fungicides are routinely applied to peach trees during the period from shuck-split through early preharvest. These cover sprays are usually applied to control peach scab, rusty spot, and anthracnose. However, results from fungicide treatments in 2010, which lacked the typical preharvest applications, indicated that these mid-season sprays may also provide control of brown rot at harvest, caused by *Monilinia fructicola*. To confirm these observations and determine the mechanism of control, a field study was conducted from 2012-2015 in an experimental peach orchard. In each year, four protectant fungicides (captan, sulfur, ziram, and thiram) were applied from shuck-split through sixth cover. Captan and thiram in 2012 and captan in 2013 and 2015 significantly reduced brown rot at harvest. Estimation of fungicide levels on fruit in 2013 to 2015, using an *in vivo* bioassay based on *M. fructicola* spore germination, showed that residual activity was the primary mechanism for control. Results also showed that captan may have a higher intrinsic efficacy than the other protectants, thereby allowing extended control through the preharvest period. These findings indicate that residual activity from mid-season applications of protectant fungicides (i) can contribute significantly to the efficacy of preharvest programs and (ii) may play an important role in reducing selection for resistance among site-specific fungicides typically applied during the preharvest period.

## Fungicide efficacy for the control of pomegranate diseases in Florida

A. KC (1), G. Vallad (2)

(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.

A statewide survey identified six economically important pathogens of Pomegranate (*Punica granatum*) in Florida. Anthracnose fruit rot and leaf spot, Botryosphaeria shoot blight and stem canker, and Piliidiella fruit rot were commonly observed throughout the growing season resulting in more than 80% fruit loss. Field trials conducted at two different locations in Central Florida evaluated Cabrio, Switch, Scala, Penncozeb, and Luna Experience for disease control on pomegranate. Fungicides were applied beginning in Early-March when > 5% flower buds were visible and reapplied every two weeks until the end of growing season in August. Disease severity was rated based on the Horsfall-Barratt scale, and values were used to calculate area under disease progress curves. Significant differences were observed between the treatments at both locations (*P* < 0.001). Cabrio, Luna Experience, and Penncozeb reduced the disease severity by 87, 84, and 67% respectively; while Switch and Scala reduced by 53 and 39% respectively compared to untreated control. Follow-up field studies are planned for 2016 to establish effective usage patterns.

## Effectiveness of fungicide treatments following the Strawberry Advisory System for control of Botrytis fruit rots in Florida

L. CORDOVA (1), A. Amiri (2), N. Peres (3)

(1) University of Florida, U.S.A.; (2) Washington State University, U.S.A.; (3) University of Florida, U.S.A.

Botrytis fruit rot (BFR) of strawberry is caused by *Botrytis cinerea*. Its management was traditionally carried out by weekly fungicide applications using multi-site fungicides in early season and single-site fungicides during bloom when the environment is conducive for disease development. Recently, many Florida growers have been following the Strawberry Advisory System (SAS) to improve timing and reduce the number of fungicide applications. Our objective was to determine the efficacy of different fungicides for BFR control when following the SAS spray timing alerts compared to preventive Calendar-based applications, and to implement specific fungicide recommendations on SAS. Field trials conducted during two seasons on a commercial farm in Plant City-FL showed that during the highest disease incidence periods, treatments with cyprodinil + fludioxonil or fluopyram following SAS and Calendar-based applications were the most effective in controlling BFR. Pyrimethanil and boscalid + pyraclostrobin were not effective due to resistance problems and are no longer recommended in the SAS. For SAS alerts during absence of bloom, a multi-site fungicide such as captan or thiram is recommended. Treatments following SAS provided good control of BFR with about half the number of fungicide applications compared to Calendar-based applications. However, the choice of the fungicide when weather conditions are highly favorable for disease development is the key for effective control.

## Interaction among leaf area index, spray volumes and fungicide doses in Asian soybean rust control

A. CHECHI (1), W. Boller (1), C. Forcelini (1), R. Roehrig (1), E. Zuchelli (1)

(1) UPF, Brazil

Soybean cultivars vary in their leaf area index (LAI), which influences the distribution of the drops generated by the spray fungicides in plant canopy. The objective of this study was to investigate the interaction among the LAI, spray volumes and fungicide doses in controlling Asian soybean rust. The tests were conducted at UPF experimental field, greenhouse and growth chamber during 2014 to 2016. The dose was varied (75%,100% and 125% of the commercial dose) and the spray volume (100,150 and 200 L/ha) at application of fungicides on soybean plants with different LAIs. An assay was also conducted with the use of simulated rainfall at different times after the fungicide application. There were three cultivars in the experimental field: BMX Ponta (high LAI); BMX Vanguarda (medium), NS 5445 (low), and only the last one in the other trial. The applications were in V7 stages (trifloxystrobin+prothioconazole), R1 and R1+20 days (azoxystrobin+benzovindiflipir) for field trials and only in V8 stage for testing in the greenhouse. The severity of the rust, the number of spores/cm², the LAI and the grain yield were quantified and analyzed by Anova and Skott-Knott test (0.05). The results showed that the spray volume and the fungicide dose should be appropriate according to the cultivar LAI. The closer the application when rain occurs, lower the control achieved by applying the fungicide. The cultivar LAI, spray volume and the fungicide dose influenced the disease control.

## Relationship between fungicide dose and Asian soybean rust control

A. CHECHI (1), C. Forcelini (2), W. Boller (2), R. Roehrig (2), E. Zuchelli (2)

(1) UPF, Brazil; (2) UPF, Brazil

The diseases affect the soybean crop yield, and the main one is the Asian soybean rust (ASR) and the control of the disease is mainly based on fungicides. The objective of this study was to verify if the use of 0 to 100% of the commercial dose of fungicide affects the control of the disease. The experiment was conducted in a growth chamber at UPF, in a completely randomized design with six replications. For this, the central leaflets of soybean plants (NS 5445 cultivar) grown in a greenhouse were collected. The solutions from 0 to 100% of the dose were prepared using two fungicides, one consisting of trifloxystrobin + prothioconazole and other azoxystrobin + benzovindiflipir. The leaflets were dipped in appropriate fungicide solutions and placed in gerboxes, where humid chambers were set up. In the next day, a suspension of *Phakopsora pachyrhizi* (50,000 spores/mL) was prepared, which was sprayed on the leaflets. The gerboxes were left in the dark for 24 hours at a temperature of 23°C. After this, they were placed on benches in a growth chamber with a photoperiod of 12 hours light/dark and constant temperature of 23°C. After 22 days incubated, the number of spores/cm² was assessed. The controls without application of fungicides showed an average of 85.4 spores/cm² (T+P) and 83.5 spores/cm² (A+B). There was a reduction in the number of spores/cm² when increases the fungicide dose. The ASR control depends on the fungicide dose in the spray solution.

## Response of Early and Late Leaf Spot Diseases on Peanut to QoI Solo Spray Programs

W. ELWAKIL (1), N. Dufault (1)

(1) University of Florida, U.S.A.

Foliar diseases including rust (*Puccinia arachidis*), early leaf spot (ELS; *Cercospora arachidicola*) and late leaf spot (LLS; *Cercosporidium personatum*) are annual problems for southeast peanut producers. Fungicides are a critical part of an integrated management program for these devastating peanut
diseases. The study objectives were to examine the efficacy of commonly used QoI fungicides for managing these diseases and to collect isolates for future pathogen population studies. Field plots of the peanut cultivar Georgia-06G were established after June 1st during 2014 and 2015 growing seasons. The fungicides azoxystrobin (Abound 2.08 SC at 18 fl oz/A) and pyraclostrobin (Headline at 9 fl oz/A) were individually applied every 12 to 17 days starting about 30 days after planting for comparison with untreated controls. Disease severity and incidence were assessed using the Florida 1-10 scale and by sampling 12 leaflets per plot. The predominant disease in 2014 was LLS and ELS in 2015. Azoxystrobin and pyraclostrobin treated plots had similar AUDPC values in 2014 and were significantly less than those of untreated plots (P ≤0.05). Mean AUDPC values were not significantly different in 2015. These results suggest that QoI fungicides have greater efficacy for LLS, however, they do not provide adequate control of ELS. Further *in vitro* investigations are ongoing in Florida to examine the population diversity of these pathogens and their QoI fungicide resistance profiles.

## Effect of fungicide timing and frequency on reducing foliar diseases and yield loss on runner and valencia type peanuts in Haiti

A. FULMER (1), T. Brenneman (1), R. Kemerait (1)

(1) University of Georgia, U.S.A.

Peanut (*Arachis hypogaea* L.) yields in Haiti are often severely reduced by peanut rust (*Puccinia arachidis*) and late leaf spot (*Cercosporidium personatum*). Two trials were conducted in 2015 to evaluate the effect of 6 fungicide regimes on disease severity and yield for two market types. Both tests used a split plot design with 4 replications with variety as whole plot and fungicide as subplot. Runner (local landrace and Cv. Georgia-06G) and valencia (local landrace and Cv. N.M. Valencia A) market types were planted in northern and central Haiti, respectively. For runners, Muscle ADV (2.3 L/ha) was applied 2-6 times starting 30-60 days after planting (DAP) with 14-28 day intervals; valencias received 1-4 sprays starting 30-45 DAP with 14-28 day intervals. There were no interactions between variety and fungicide regime. At each location, disease severity was similar between varieties, but Georgia-06G and the local valencia yielded 2500 and 430 kg/ha more, respectively. For valencias, at least 2 sprays were required to reduce severity and increase yield; however, 3 or more sprays gave the highest yields. For runners, 3 and 4 sprays were required to reduce rust and leafspot, respectively, but 6 sprays had the least disease and the only statistically different yield. Compared to untreated plots, the best treatments increased yields by 68% for both market types. These results are a significant step toward developing management recommendations for growers in Haiti.

## Fungicides for management of *Sclerotium rolfsii* and enhanced overwintering survival of stevia

A. KOEHLER (1), H. Shew (1)

(1) NCSU, U.S.A.

Stevia (*Stevia rebaudiana*) is an herbaceous perennial under evaluation as a new crop in NC. Stem rot caused by *Sclerotium rolfsii* results in wilting, rapidly followed by root and stem necrosis. Currently there are no fungicides labeled for use on stevia, which will limit expansion of commercial production. Initial fungicide efficacy trials were established in July 2014 in naturally infested soil in Kinston, NC. Treatments included azoxystrobin, flutolanil, and tebuconazole applied at 3 timings. This trial was repeated in May and July of 2015 in adjacent field space. Stand counts were taken to determine disease incidence and overwintering survival of stevia crowns. All fungicides reduced incidence of stem rot compared to the control. In 2015, azoxystrobin was correlated with enhanced overwintering survival (mean survival rate, 66%) compared to other fungicide treatments (19%) and the untreated control (16%). In 2015, a second field site was established in Rocky Mount, NC to evaluate the effect of late season applications of azoxystrobin and pyraclostrobin. Both fungicides resulted in higher root weights and greater new shoot production compared to the control in spring 2016. Final stand counts of all treatments will be taken in spring 2016 to establish overwintering survival rates. Successful overwintering of stevia crowns directly impacts the profitability of second and third year harvests and the likelihood of long term establishment of stevia in NC.

## Timing of fungicide applications is critical for blackleg control in canola

L. DEL RIO MENDOZA (1), S. Ruud (1), S. Mansouripour (1)

(1) North Dakota State University, U.S.A.

Blackleg, caused by *Leptosphaeria maculans*, is an important disease affecting rapeseed/canola (*Brassica napus*) production worldwide. The genetic resistance currently available in most commercial cultivars is ineffective against strains of blackleg that have become prevalent in recent years and because of this, growers are relying more frequently on fungicide use to manage this disease. To optimize fungicide use, greenhouse studies evaluated the efficacy of different timings of application of azoxystrobin, pyraclostrobin, and fluxapyroxad on blackleg severity. These active ingredients, present in four of the six fungicides registered to manage blackleg in North Dakota, were applied two days before and 0, 2, 4, 8, or 16 days after seedlings of cv. Westar were inoculated with an *L. maculans* spore suspension. Disease severity was evaluated 12 days after inoculation and before harvest. For each fungicide, trials were conducted separately using a randomized complete block design. Each study was replicated and conducted three times. All three fungicides were most effective when applied within eight days from inoculation and lost their efficacy when applied at a later time. This information highlights the need for early protection of seedlings as well as for the development of disease warning systems that could identify help growers optimize spraying schedules.

## Evaluation of fungicide efficacy and cultivar susceptibility for the management of anthracose on celery

M. MCDONALD (1)

(1) University of Guelph, Canada

Anthracnose on celery, also called celery leaf curl, is caused by *Colletotrichum fioriniae*. The disease was first identified in Ontario, Canada, in 2011 and has been increasing in severity. Field trials were conducted to determine if there were differences in cultivar susceptibility and to identify effective fungicides. The celery cultivars were: TZ6010, TZ0295, TZ6200, TZ6029, TZ9779, Nero, Sabroso and Plato. Cv. TZ 6200 was used for the fungicide trial. Seedlings were transplanted on 24 June into 'muck' soil (organic matter ~70%, pH 6.5) in the Holland Marsh, Ontario. Each replicate consisted of 3 rows, 55 cm apart and 5 m in length. The trials were inoculated on 18 August with a suspension of $1 \times 10^5$ conidia ml$^{-1}$, and harvested on 13 October. The fungicides evaluated were Omega (fluazinam 40.0%), Bravo (chlorothalonil 50%), and Flint (trifloxystrobin 50%). Fungicides were applied on 31 July, 13 August and 8 September, 2015. Disease incidence and marketable yield were assessed. Cultivars Plato, TZ 9779, 6200 and 0295 had high disease incidence (86, 66, 66, and 67%) compared to less susceptible cv.'s Sabroso (24%) and TZ 6010 (27%). Fungicides Flint and Bravo had low disease (34, 45%), compared to the untreated check (83%). Increased disease incidence in September was related to a decrease in the percent of marketable yield by weight ($r^2=0.45$).

## Optimizing the systemicity of triazole formulations by enhancing leaf penetration

G. POON (1), C. Irwin (2), J. Dinglasan (2), N. Loukine (1)

(1) Vive Crop Protection, Canada; (2) Vive Crop Protection, Canada

The systemicity of a fungicide plays a vital role in its performance. While the ability of a fungicide to penetrate the leaf cuticle and translocate through plant tissue is active specific, inert formulation ingredients can optimize these processes on target crops. Triazoles and strobilurins are broad spectrum fungicides with known systemicity; however, in lab testing only a small fraction of applied active ingredient was able to penetrate a synthetic membrane that mimics a leaf cuticle. Fungicide formulations that are optimized for membrane penetration may have enhanced disease control over existing formulations. Laboratory trials were conducted using a Franz cell fitted with a synthetic membrane to identify experimental formulations that may have better leaf cuticle penetration than the commercial product. Field trials were conducted in 2015 to evaluate the efficacy of experimental tebuconazole formulations on powdery mildew in acorn squash. All tebuconazole treatments were applied weekly at label rate following disease onset for a total of