# Alternatives for the management of coffee corky-root disease: Search for tolerance and antagonistic fungi

Dinorah Lima-Rivera, Alejandro Salinas-Castro, Gerardo Gutierrez-García, Andrés Rivera-Fernández, Carlos Cerdan, Luc Villain, Damaris Desgareennes, Daniel Lopez-Lima

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## To cite this version:

Dinorah Lima-Rivera, Alejandro Salinas-Castro, Gerardo Gutierrez-García, Andrés Rivera-Fernández, Carlos Cerdan, et al.. Alternatives for the management of coffee corky-root disease: Search for tolerance and antagonistic fungi. Tropical and Subtropical Agroecosystems, 2025, 28 (2), pp.083. 10.56369/tsaes.6030. hal-05428476

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# ALTERNATIVES FOR THE MANAGEMENT OF COFFEE CORKY-ROOT DISEASE: SEARCH FOR TOLERANCE AND ANTAGONISTIC FUNGI †

## [ALTERNATIVAS PARA EL MANEJO DE LA CORCHOSIS DE LA RAIZ DEL CAFETO: BUSQUEDA DE TOLERANCIA Y HONGOS ANTAGONISTAS]

Dinorah Lima-Rivera¹, Alejandro Salinas-Castro²,
Gerardo Gutierrez-García¹, Andrés Rivera-Fernández¹, Carlos Cerdán¹,
Luc Villain³,⁴, Damaris Desgarennes⁵ and Daniel López-Lima¹*

¹ Universidad Veracruzana, Facultad de Ciencias Agrícolas, 91090 Xalapa, Veracruz, México. Email: danielopez@uv.mx

² Universidad Veracruzana, Centro de Investigación en Micología Aplicada, 91010 Xalapa, Veracruz, México. Email: asalinas@uv.mx

³ CIRAD, UMR DIADE, F-34394 Montpellier, France. Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR DIADE, 34398 Montpellier, France.

⁴ Diversité, Adaptation et Développement des Plantes (DIADE), Université de Montpellier, Pole Agriculture Environnement Biodiversité, IRD, CIRAD, 34394 Montpellier, France.

⁵ Red de Biodiversidad y Sistemática, Instituto de Ecología A.C., 91073 Xalapa, Veracruz, Mexico. Email: damaris.desgarennes@inecol.mx

*Corresponding author

## SUMMARY

**Background:** The root-knot nematode *Meloidogyne paranaensis* is one of the main phytosanitary problems in coffee crops, as it causes premature death of plants. In localities in central Veracruz, *Coffea canephora* plants tolerant to this nematode have been detected, which can be used as rootstocks in infested areas. **Objective:** To select plants that show tolerance in the field and to isolate and identify fungi associated with the rhizosphere of these coffee plants. **Methodology:** Plants infested with nematodes were selected and root samples were taken to quantify population density and corroborate the species. Fungi were isolated and identified from the rhizosphere soil. Five fungi were selected to observe the *in vitro* infection process on *M. paranaensis* eggs.

**Results:** 18 plants that showed tolerance to *M. paranaensis* were selected. Twenty strains of fungi belonging to 7 genera and 16 species were obtained. The genera *Penicillium* and *Fusarium* were the most frequent. *Clonostachys rogersoniana* was the only fungus that showed parasitism against *M. paranaensis* eggs *in vitro*.

**Implications:** The 18 selected plants show tolerance in the field, and it is necessary to conduct tests with controlled inoculations of *M. paranaensis* with clones and progeny. Tests on the nematophagous capacity of *C. rogersoniana* should continue. **Conclusion:** In the study area there are plants with tolerance to *M. paranaensis* and they can be used as rootstocks. The fungus *C. rogersoniana* is capable of infesting *M. paranaensis* eggs and is a potential biological control agent for this nematode.

**Key words:** *Meloidogyne paranaensis*; root-knot nematode; tolerant plants; nematophagous fungi; soil borne diseases.

## RESUMEN

**Antecedentes:** El nematodo agallador *Meloidogyne paranaensis* es uno de los principales problemas fitosanitarios del cultivo de café ya que provoca la muerte prematura de las plantas. En localidades del centro

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<footnote>† Submitted November 23, 2024 – Accepted January 14, 2025. http://doi.org/10.56369/tsaes.6030</footnote>
de Veracruz se ha detectado plantas de *Coffea canephora* tolerantes a este nematodo, que pueden utilizarse como portainjertos en áreas infestadas. **Objetivo:** Seleccionar plantas que muestren tolerancia en campo y aislar e identificar hongos asociados a la rizosfera de estos cafetos. **Metodología:** Se seleccionaron plantas infestadas con nematodos y se tomaron muestras de raíz para cuantificar densidad de población y corroborar la especie. Se aisaron e identificaron hongos del suelo rizosférico. Se seleccionaron cinco hongos para observar el proceso de infección *in vitro* sobre huevos de *M. paranaensis*. **Resultados:** Se seleccionaron 18 plantas que presentaron tolerancia a *M. paranaensis*. Se obtuvieron 20 cepas de hongos pertenecientes a 7 géneros y 16 especies. Los géneros *Penicillium* y *Fusarium* fueron los más frecuentes. *Clonostachys rogersoniana* fue el único hongo que mostró parasitismo contra los huevos de *M. paranaensis in vitro*. **Implicaciones:** Las 18 plantas seleccionadas muestran tolerancia en campo y es necesario realizar pruebas con inoculaciones controladas de *M. paranaensis* con clones y progenie. Se debe continuar con las pruebas sobre la capacidad nematófaga de *C. rogersoniana*.
**Conclusión:** En la zona de estudio hay plantas con tolerancia a *M. paranaensis* y que tienen posibilidad de utilizarse como portainjertos. El hongo *C. rogersoniana* es capaz de infestar huevos de *M. paranaensis*, y es un potencial agente de control biológico de este nematodo.

**Palabras clave:** *Meloidogyne paranaensis*; nematodos agallador; plantas tolerantes; hongos nematofagos; plagas del suelo.

INTRODUCTION

Coffee growing is one of the main economic activities for more than 60 countries in Latin America, Africa, and Asia. Approximately 120 million people depend directly or indirectly on its production, Brazil, Vietnam, Indonesia, and Colombia are the main producers (FAO, 2024). Mexico is the thirteenth largest producer with more than 180 thousand tons of green coffee harvested per year, by more than 500 thousand producers in 48 municipalities (FAO, 2024, SIAP, 2024). In 2023, almost 700 thousand hectares were planted, with the states of Chiapas and Veracruz being the main producing states, contributing 34.8 and 20.6% of the total national volume (SIAP, 2024). Currently one of the main phytosanitary problems affecting coffee production in America are the root-knot nematodes (Villain *et al.*, 2013). These nematodes cause considerable economic losses by affecting the root and preventing the proper development of the plant (Elling, 2013; Villain *et al.*, 2013). In particular, *Meloidogyne paranaensis* is the most harmful species for coffee plants as it is involved in the development of the disease known as coffee corky-root, which begins with the infestation of the nematode and gradually worsens with the subsequent arrival of phytopathogenic and opportunistic fungi and bacteria (López-Lima *et al.*, 2020; Lamelas *et al.*, 2020). Infested plants have reduced longevity, forcing growers to constantly replace plantations (Lima-Rivera *et al.*, 2024). In Mexico, there are records of damage caused by coffee corky-root disease since the 1960s, however, until the 2000s it was only distributed in small areas of the states of Veracruz and Chiapas (Vázquez, 1963; López-Lima, 2021). Since 2013, with the massive replacement of coffee plants due to the epidemic of yellow leaf rust

(*Hemileia vastatrix*) (Chort and Berk, 2024), coffee corky-root disease was widely distributed because plants infested with *M. paranaensis* were planted in fields free of this nematode (López-Lima, 2021). To date, *M. paranaensis* is the only root-knot nematode species associated with the development of coffee corky-root disease in Mexico (López-Lima *et al.*, 2015; Alcasio-Rangel *et al.*, 2017). To mitigate this problem, *Coffea canephora* (robusta) rootstocks are used because the plants of this species have a more vigorous root system, which increases the longevity of the plants for some years. However, they are susceptible to infestation by *M. paranaensis* and represent a temporary solution, since most of the grafted plants end up dying prematurely (Fatobene *et al.*, 2019). The oldest records of the presence of coffee corky-root disease in Mexico come from the central mountainous area of Veracruz, in the municipalities of Tlaltetela and Totutla (Vázquez, 1963). In some localities of the Totutla municipality, farmers began planting robusta coffee trees in the 1980s in order to mitigate the effects of disease. In previous surveys, it was detected that some robusta coffee plants have shown good levels of development, vigor, and longevity despite being affected by coffee corky-root disease. The study of these plants could generate alternatives to increase the longevity of plantations in infested areas. Since in Mexico there are no coffee varieties with tolerance to *M. paranaensis* that are used as rootstocks and no studies have been conducted on microorganisms antagonistic to *M. paranaensis*, the objective of this work was to carry out a first selection of plants that show tolerance in the field and to isolate and identify fungi associated with the rhizosphere of these coffee plants with the potential to be used as biological control agents for *M. paranaensis*.
# MATERIALS AND METHODS

## Study site and plant selection

Sampling was conducted in a variable width transect through the coffee plantation area of the towns of Mata Obscura and Mata de Indio in the municipality of Totutla. The plant selection criteria were based on the age (10 years minimum), presence of coffee corky-root symptoms and plant vigor (Cerda-Ocaranza et al., 2023). Root and rhizospheric soil samples were taken from the selected plants, which were placed in polypropylene bags and transported to the laboratory. The roots were washed with running water and dried with absorbent paper to obtain the fresh weight, observe the formation of galls or corky-root symptoms and assign a number on the galling scale (Coyne and Rose, 2014). Subsequently, the roots were cut into 1-2 cm fragments and 20 g were taken to extract nematode eggs and second stage juveniles (J2) by sodium hypochlorite grinding, sieving and centrifugation technique (Carneiro et al., 2004). A 10 mL suspension of eggs and J2 was obtained from which three one mL aliquots were taken and counted in a Sedgwick-Rafter chamber under an optical microscope at 100X. Population density (eggs and $J2 \text{ g root}^{-1}$) was calculated by dividing the average nematode count by the weight of the root processed for extraction (average nematode count/weight of processed root). The identification of the nematodes obtained from the roots was corroborated by the *M. paranaensis* species-specific SCAR marker (Randig et al., 2002).

## Isolation and identification of fungi

Isolation of fungi from rhizospheric soil was performed by the agar plate dilution method (Carrion et al., 2021). Five g of soil were taken from each sample and diluted in 10 mL of sterile water. The solution was vortexed for five minutes. 100 µL of this solution was then inoculated into Petri dishes (80 mm diameter) with potato dextrose agar (PDA) culture medium. The Petri dishes were incubated at 22 °C and observed daily. Fungal colonies that grew on the agar plates were separated into new PDA dishes until pure cultures were obtained.

From the pure cultures of each fungus, DNA was extracted using the Kit Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit ®. A 500 bp molecular marker that encompasses the Internal Transcriber Spacer (ITS) 1 and the ITS2 molecular markers, were used and amplified by PCR (Schoch

et al., 2012). The PCR products were analyzed on a 1.2 % agarose gel; and the DNA was purified and sent to Macrogen INC for sequencing. The obtained sequences were edited in the FinchTV 1.4.0 program and compared by BLAST analysis to the database of the National Center for Biotechnology Information (NCBI).

## Fungal infection process on *M. paranaensis* eggs

To make a first selection of strains with nematicidal potential, an *in vitro* test was made exposing viable eggs of *M. paranaensis* to five fungi, selected based on their identification and the genus history of use as biological control agents. Nematode eggs were extracted by the method of grinding in sodium hypochlorite, sieving and centrifuging from *C. arabica* roots previously infested with *M. paranaensis* (Carneiro et al., 2004). The extraction solution was quantified in a Sedgwick-Rafter counting chamber and adjusted to 1000 eggs mL⁻¹. Subsequently, 100 µL of the *M. paranaensis* egg suspension was placed in a Petri dish with a previously prepared agar-water plate. In parallel, a suspension of 1x10⁶ spores mL⁻¹ of each selected fungus was made with sterile distilled water and 100 µL of this solution was placed on the eggs previously deposited on the agar-water plate. To observe the infection process, spore germination was stopped every 24 hours on five occasions by adding a drop of lactophenol blue. The eggs were then placed on a slide to observe under a microscope characteristics such as germination or penetration of the hyphae into the cuticle of the eggs. This procedure was performed in quintuplicate for each fungal strain and for each hour of sampling. Likewise, a group of eggs were kept without inoculation as an untreated control (N=150).

# RESULTS AND DISCUSSION

## *Coffea canephora* plants with tolerance to *Meloidogyne paranaensis* selected in *situ*

Eighteen *C. canephora* plants distributed in 9 farms were selected (Table 1). The plants showed good development with abundant shoot generation despite the infestation (Figure 1A). Young roots exhibited gall formation (Figure 1B), and woody roots showed symptoms of corky-root such as thickening of the cortical tissue, necrosis (Figure 1C) and the presence of numerous *Meloidogyne* female embedded in the tissue (Figure 1D). The galling level of the 18 plants was light to medium, at levels 2 and 3 on the scale, and the averaged population density of 79.3 eggs and $J2 \text{ g root}^{-1}$
(Table 1). The use of nematode-tolerant plants is one of the most effective nematode management strategies, since, in most cases, these plants maintain their level of productivity even when they are infested (Shigueoka et al., 2016). Likewise, microorganisms present in the rhizosphere can function as regulators of pest populations, giving the plant a chance of overcoming the infestation (Topalovic et al., 2020). In Mexico there are no *C. canephora* selections or cultivars with proven resistance or tolerance to *M. paranaensis* that can

be used systematically as rootstocks. The search for tolerant plants in infested areas is a commonly strategy, however, further studies are needed with the plants selected in this work, in order to determine the origin of the tolerance observed in the field, which may be due, in addition to genetic factors, to the edaphoclimatic conditions of the site or the presence of microorganisms antagonistic to nematodes in the rhizosphere (Sera et al., 2010; Lima-Salgado et al., 2014).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** *Coffea canephora* plant infested with *Meloidogyne paranaensis*. A) Aerial aspect of the infested plant showing vigor and development, B) secondary roots with thickening caused by *M. paranaensis*, C) Woody root with symptoms of corchosis, D) *M. paranaensis* females embedded in the corky tissue 40X. Barr = 1 cm.</figcaption></figure>
<table><caption>Table 1. Geographic data of sampling sites, galling level, and population density of *Meloidogyne paranaensis* of the selected plants (average of two plants).</caption><thead><tr><th>Locality</th><th>Site</th><th>Location</th><th>Altitude (masl)</th><th>Galling index</th><th>Population density eggs and J2 g root<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="4">Mata oscura</td><td>1</td><td>19°13'17.0"N; 96°51'08.3"W</td><td>947</td><td>2</td><td>58</td></tr><tr><td>2</td><td>19°14'03.9"N; 96°50'33.2"W</td><td>896</td><td>2.5</td><td>88.5</td></tr><tr><td>3</td><td>19°13'30.9"N; 96°51'07.4"W</td><td>939</td><td>3</td><td>106</td></tr><tr><td>4</td><td>19°13'20.4"N; 96°51'16.2"W</td><td>957</td><td>2.5</td><td>84</td></tr><tr><td rowspan="5">Mata de Indio</td><td>5</td><td>19°14'01.0"N; 96°50'36.2"W</td><td>898</td><td>2</td><td>74.5</td></tr><tr><td>6</td><td>19°13'48.1"N; 96°50'52.6"W</td><td>922</td><td>2</td><td>74.5</td></tr><tr><td>7</td><td>19°13'37.6"N; 96°50'46.9"W</td><td>921</td><td>2.5</td><td>79.5</td></tr><tr><td>8</td><td>19°14'02.7"N; 96°50'26.6"W</td><td>889</td><td>2</td><td>73</td></tr><tr><td>9</td><td>19°13'57.8"N; 96°50'32.2"W</td><td>896</td><td>2</td><td>76</td></tr></tbody></table>

## Native fungi associated with the rhizosphere of *Coffea canephora* with corky-root disease

Twenty fungal strains belonging to 7 genera and 16 species were isolated from the sampled sites. The genus *Penicillium* was the most frequent with seven isolates followed by *Fusarium* with six isolates. The most frequent species was *Fusarium oxysporum* with four isolates (Table 2).

Many fungi have been reported associated with coffee plants, the main fungal families recorded are Aspergillaceae and Netriaceae (Lu et al., 2022), which coincides with the results of this work where the most frequent species belong to these families.

Likewise, in a study of the rhizosphere microbiome of coffee plants, the genera *Fusarium*, *Trichoderma*, *Aspergillus* and *Rhizophagus* were the most abundant (de Sousa et al., 2022). In another study, fungi were isolated directly from coffee corky-root tissues, and 49 isolates were obtained, *Fusarium* and *Penicillium* were the main genera with a relative abundance of 65 and 10% respectively (Lopez-Lima et al., 2020). Many of the fungi found in this work have reports of biological activity against pest nematodes, for example, *Trichoderma spirale* has been reported parasitizing *Radopholus similis*, *Meloidogyne exigua* and *Meloidogyne incognita* (Varela-Benavides et al., 2017). *Penicillium* species are

<table><caption>Table 2. Molecular identification of fungal species isolated from coffee rhizosphere of plants with corky-root disease.</caption><thead><tr><th>Locality</th><th>Site</th><th>Fungi species<br>molecular diagnosis</th><th>NCBI accession number</th><th>% identity</th></tr></thead><tbody><tr><td rowspan="6">Mata oscura</td><td rowspan="3">1</td><td>Penicillium tanzanicum</td><td>NR_158820.1</td><td>100%</td></tr><tr><td>Penicillium glaucoroseum</td><td>MT530148.1</td><td>99.8%</td></tr><tr><td>Fusarium proliferatum</td><td>OQ363325.1</td><td>100%</td></tr><tr><td>2</td><td>Fusarium oxysporum</td><td>PP596543.1</td><td>100%</td></tr><tr><td>3</td><td>Fusarium oxysporum</td><td>OQ683854.1</td><td>100%</td></tr><tr><td>4</td><td>Penicillium tealli</td><td>OP101644.1</td><td>100%</td></tr><tr><td rowspan="9">Mata de Indio</td><td rowspan="4">5</td><td>Trichoderma kaningiopsis</td><td>MT520626.1</td><td>100%</td></tr><tr><td>Penicillium jiangxiense</td><td>KP204361.1</td><td>100%</td></tr><tr><td>Trichoderma spirale</td><td>KF624809.1</td><td>100%</td></tr><tr><td>Gleotinia temulenta</td><td>KT876537.1</td><td>100%</td></tr><tr><td rowspan="2">6</td><td>Penicillium kloeckeri</td><td>KT385771.1</td><td>98.9%</td></tr><tr><td>Clonostachys rogersoniana</td><td>MH421856.1</td><td>100%</td></tr><tr><td rowspan="3">7</td><td>Penicillium glaucoroseum</td><td>MT530148.1</td><td>100%</td></tr><tr><td>Aspergillus westerdijkiale</td><td>MT378420.1</td><td>100%</td></tr><tr><td>Fusarium oxysporum</td><td>PP596543.1</td><td>100%</td></tr><tr><td rowspan="4">8</td><td>Metarhizium marquandii</td><td>MH483861.1</td><td>100%</td></tr><tr><td>Fusarium graminearum</td><td>PP620750.1</td><td>100%</td></tr><tr><td>Fusarium oxysporum</td><td>PP596543.1</td><td>100%</td></tr><tr><td>Penicillium stratisporum</td><td>OQ332398.1</td><td>100%</td></tr><tr><td>9</td><td>Metarhizium robertsii</td><td>MT378171.1</td><td>99.8%</td></tr></tbody></table>
widely used as biological control agents, in particular nematicidal activity has been reported in *P. commune* and *P. chrysogenum* species (Sikandar *et al.*, 2020; Nguyen *et al.*, 2021). Non-pathogenic strains of *Fusarium oxysporum* have been found parasitizing *R. similis*, *Pratylenchus goodeyi* and *M. incognita* (Dababat and Sikora, 2007; Van Dessel *et al.*, 2011). However, pathogenic strains of *F. oxysporum* are commonly associated with coffee corky-root and this fungus is considered one of the main causative agents of this disease (López-Lima *et al.*, 2020), which may explain the frequency isolation of *F. oxysporum* in this work. On the other hand, the fungus *Metarhizium robertsii* has been reported for use in biological control of fungi and insects, for example, it is antagonistic to the phytopathogenic fungus *Cochliobolus heterostrophus* and pathogenic to the insect pest *Spodoptera frugiperda* (Flonc *et al.*, 2021; Ahmad *et al.*, 2022).

**In vitro fungal infection process on M. paranaensis**

The eggs infection process was conducted with the
*Aspergillus westerdijkiae*, *Clonostachys*

*rogersoniana*, *Gleotinia temulenta*, *Penicillium glaucoroseum* and *Penicillium striatisporum* strains. For *A. westerdijkiae*, no spore germination was observed in the first 24 and 48 h, at 72 and 96 h the development of hyphae was observed on the outside of the eggs and at 120 h the presence of hyphae surrounding the cuticle of the eggs was observed, but with no evidence of penetration (Figure 2A). For *C. rogersoniana*, no spore germination was observed in the first 24 h, at 48 h spore germination was observed on the cuticle of the eggs, at 72 h the eggs were found surrounded by mycelium and for 96 and 120 h the mycelium was observed inside the eggs and the degradation of the content (Figure 2B). In the eggs treated with *G. temulenta*, no spore germination or mycelium was observed in any of the observations (Figure 2C). No germination of *P. glaucoroseum* spores was observed at 24 and 48 h. At 72 and 96 h, hyphal development was observed and at 120 h abundant mycelial development was seen around the eggs with no evidence of penetration (Figure 2D). Spores of *P. stratisporum* were observed attached to the eggs, but no evidence of germination during the experiment (Figure 2E). Eggs without the application of fungus remained without mycelial

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Infection process of fungi isolated from the rhizosphere of *Coffea canephora* on *Meloidogyne paranaensis* eggs at 120 h after inoculation. A) *Aspergillus westerdijkiae*, B) *Clonostachis rogersoniana*, C) *Gleotinia temulenta*, D) *Penicillium glaucoroseum*, E) *Penicillium striatisporum* and F) untreated control. 400 X. Bar = 30 µm.</figcaption></figure>
development or alterations during the experiment (Figure 2F). *Aspergillus* strains have demonstrated nematicidal activity, for example, *Aspergillus* sp. strain SPH2 kills 23.7% of *M. javanica* juveniles under *in vitro* conditions (Morales-Sánchez *et al.*, 2021). *Aspergillus fumigatus* produces 2-furoic acid in liquid fermentation and exposure to the culture broth causes mortality of J2 juveniles of *M. incognita* at a dose of 37.75 µg/mL (Wang *et al.*, 2022). Likewise, the fermentation filtrate of *Aspergillus japonicus* kills 100% of the J2 juveniles of *M. incognita* within 24 h and inhibits the eggs hatching (He *et al.*, 2020). It is necessary to evaluate the nematicidal activity of the fermentation metabolites of *A. westerdijkiae*, since in this work with the direct application of spores no penetration of mycelium into the eggs was observed. The *Clonostachys* genus is widely recognized as a biological control agent for its production of secondary metabolites that have pharmaceutical or agrochemical applications (Han *et al.*, 2020). In particular, *C. rogersoniana* is known to produce verticillins, which are dimeric epipolythiodixopiperazines with antimicrobial activity (Guo *et al.*, 2017). *Clonostachys rogersoniana* was the only fungus that showed egg degradation, tests are needed to determine its potential use as a biological control agent for nematodes.

The nematicidal potential of *Penicillium* strains has been previously demonstrated. For example, the fermentation broth of *Penicillium chrysogenum* causes up to 97% mortality of J2 juveniles and 94% inhibition of hatching after 72 h of exposure to *M. incognita* eggs (Sikandar *et al.*, 2020). The nematicidal activity of *Penicillium* is related to secondary metabolites production, as studied in *Penicillium commune*, which produces cyclopiazonic acid and has activity against second-stage juveniles of *M. incognita*, *M. hapla* and *M. arearia* (Nguyen *et al.*, 2021). The two *Penicillium* species evaluated in this work did not show penetration of the egg cuticle by hyphae, however, it is necessary to study the effect of their fermentation metabolites.

The data obtained in this work do not allow us to determine whether the tolerance shown by the *C. canephora* plants at the study site is due to the presence of certain fungal species, since no fungal species was detected in common for all sites. It is necessary to perform nematicidal activity tests with all the strains isolated individually and in consortia by isolation sites in order to determine whether these influence the tolerance to *M. paranaensis* shown by the plants.

## CONCLUSION

The use of different alternatives to mitigate the *M. paranaensis* population is the best strategy to reduce the effects of coffee corky-root disease. The sowing of plants with tolerant rootstocks can be complemented with the application of antagonistic organisms and even with the application of synthetic nematicides at crucial moments such as the establishment of new plants. This work shows that in the study area there are plants that can tolerate the infestation by *M. paranaensis* and that have the possibility of being used as rootstocks. It is necessary to conduct studies with clones and progeny of the selected plants in controlled inoculations with different population densities of *M. paranaensis* to determine their tolerance level. Likewise, the evaluation of the fungus *C. rogersoniana* should continue since this work demonstrated its ability to infest *M. paranaensis* eggs and be used as a biological control agent for this nematode.

## Acknowledgements

The first author thanks the National Council of Humanities, Sciences and Technologies (CONAHCYT) for the scholarship granted to conduct her postgraduate studies (PhD in Agricultural Sciences) at the Universidad Veracruzana (UV).

**Funding.** No financial support was received to conduct this work.

**Conflict of interest.** The authors declare that they have no competing interests.

**Compliance with ethical standards.** The authors confirm that this investigation was conducted under the current ethical procedures. No humans or animals were used in the studies of this article.

**Data availability.** Data is available with the corresponding author upon request.

**Author contribution statement (CRediT).** **D. Lima-Rivera** – Writing – review and editing, Investigation, Methodology. **D. Desgarennes** – Methodology, Validation. **G. Gutierrez-García** – Investigation, Methodology. **A. Rivera-Fernández** – Conceptualization, Investigation, Validation, Supervision. **C. Cerdán** – Conceptualization, Validation, Supervision. **A. Salinas-Castro** – Conceptualization, Validation, Supervision. **L. Villain** – Conceptualization, Validation, Supervision. **D. López-Lima** –
Investigation, Conceptualization, Investigation, Writing-review and editing, Supervision.

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