Abstract:
The present invention provides a novel isolated and purified  Harknessia lythrii . It further provides a mycoherbicidal composition that is effective in controlling purple loosestrife, and methods for controlling purple loosestrife.

Description:
PRIORITY OF INVENTION  
       [0001]    This application claims priority to U.S. application No. 09/240,160, filed Jan. 29, 1999 which application is incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    Purple loosestrife ( Lythrum salicaria ) is an introduced plant that has escaped cultivation to become an aggressive invasive weed that is spreading rapidly throughout North America. It forms uniform stands that replace diversified native flora, including important waterfowl food plants. Because its stands are dense, it destroys wetlands by reducing water flow and drying out the soil. Many wetlands in North America have major infestations of purple loosestrife. It has no natural enemies on the continent, neither native herbivores nor pathogens, that presently suppress invasive populations of purple loosestrife. Further, its growth is so dense that most wetland wildlife cannot use it as habitat. Moreover, it is not a preferred food for native animals in North America.  
           [0003]    A number of characteristics have enabled purple loosestrife to become a problem. A single, mature plant can produce 2.5-million seeds annually. Established plants grow more than 2 meters tall with 30-50 stems forming wide crowns that dominate a plant canopy. Also, a strong rootstock serves as a storage organ providing resources for spring growth and regrowth if the above-ground shoots are destroyed. R. F. Nyvall,  Mycologia,  87:501-506 (1995).  
           [0004]    The U.S. Department of Natural Resources as well as most states in the U.S. have declared it a noxious weed and require that it be controlled. It is also on the Noxious Weed List in Alberta, Manitoba, and numerous municipalities in Canada. Governmental agencies have been trying to control the spread of purple loosestrife by various means including cutting it, pulling it out, spraying it with herbicides and burning it. Some of the control methods make the situation worse by killing surrounding plants so the purple loosestrife seedlings have no competition. For example, the seemingly obvious solution of pulling it up by the roots does not work. The whole root mass must be removed, causing extensive disturbance to the soil, and creating the very habitat in which this plant thrives. Also, all of the plant must be removed, because it can regenerate from a tiny piece of crown, stalk or leaf buds. Chemical herbicides such as ROUNDUP® or RODEO® are not desirable because they kill essentially all the wetland plants since they are not selective for purple loosestrife.  
           [0005]    In addition to there not being acceptable chemical herbicides for purple loosestrife, environmental concerns make biological control a potentially attractive alternative to traditional methods of weed control. Biological control of purple loosestrife began in Germany in the 1960&#39;s. It was found that certain species of beetles ( Galerucella calmariensis, G. pusilla , and  Hylobius transversovittatus ) could help control the spread of the weed. In 1991, the Canadian government approved the introduction of these predators of purple loosestrife as biological control agents. In 1992, the United States Department of Agriculture approved the release of these beetles as biological control agents in the U.S.  
           [0006]    Unfortunately, it takes quite a long time (3-5 years) for these beetles to become established in an area, if they become established at all. Further, even if they become established in an area, they may not significantly impact purple loosestrife for at least 2-3 years because the plant is so resilient. Moreover, the beetles do not always kill the crown of the plant, so they may not consistently prevent seed production by the plants. The seed bank of purple loosestrife is viable for approximately nine years.  
           [0007]    To date, no mycoherbicides have been developed from pathogens of purple loosestrife either in Europe or North America. In fact, disease symptoms are rarely observed in Europe and no pathogens have been isolated from purple loosestrife in Europe with the exception of a nematode Meloidogyne sp. reported to feed on roots in the former USSR. R. F. Nyvall,  Mycologia,  87:501-506 (1995).  
           [0008]    Thus, there remains a continuing need for a means to safely and effectively control the spread of purple loosestrife. There is further a long-felt, unresolved need to produce an herbicidal composition that can be sprayed, or similarly administered, onto purple loosestrife or other noxious weeds to selectively control the weeds in environments with mixed populations of useful plants, such as in a protected wetland.  
         SUMMARY OF THE INVENTION  
         [0009]    The present invention provides an isolated and purified  Harknessia lythrii  fungus. This fungus can be isolated from cultivated plant material of the genus Lythrum and has been deposited with the ATCC with accession number PTA-2756.  
           [0010]    The present invention also provides herbicidal composition containing an effective amount of an isolated and purified  Harknessia lythrii  and/or  Coniella frugarei  that effects control of purple loosestrife ( Lythrum salicaria ), and a carrier. The  Coniella frugarei  has been deposited with the ATCC with accession number PTA-2757. The carrier in the herbicidal composition may be diatomaceous earth, alginate or clay, or may be a liquid carrier. The carrier may be an adjuvant, or may contain an adjuvant. Such an adjuvant may be an effective plant-tissue penetrating adjuvant, and may be water-miscible or water-dispersable. For example, the adjuvant may be a methylated seed oil. The  Harknessia lythrii  in the herbicidal composition may be in the spore form, and may, for example, be present in the range of about 1×10 2  to about 1×10 12  spores per ml, in the range of about 1×10 4  to about 1×10 9  spores per ml, or in the range of about 1×10 5  to about 1×10 8  spores per ml. The herbicidal composition may also contain an effective amount of at least one additional fungus, such as  Coniella frugarei , which may be in the spore form, to form a fungal composite. The total quantity of spores in the fungal composite may be in the ranges given above. The herbicidal composition may also contain a germination activator, such as a monosaccharide, disaccharide, polysaccharide, amino acid, peptide, peptone, protein, or inorganic salt.  
           [0011]    The present invention further provides a method for controlling purple loosestrife ( Lythrum salicaria ) by applying an effective amount of a herbicidal composition onto a target plant or onto the situs of a target plant, wherein the herbicidal composition contains isolated and purified  Harknessia lythrii  and a carrier, as described above. It may further contain an effective amount of at least one additional fungus to form a fungal composite, as described above. The herbicidal composition is applied at least once, and the target plant may be controlled for multiple growing seasons., wherein the fungal composite is in the spore form. The method may also be practiced by additionally applying a plant stressing agent to the target plant. The plant stressing agent may be an insect, such as  Galerucella calmariensis  or  Hylobius transversovittatus   
       
    
    
     DETAILED DESCRIPTION OF THE FIGURES  
       [0012]    FIGS.  1 - 14 .  Harknessia lythrii.    
         [0013]    [0013]FIG. 1. Conidiomata produced on PDA.  
         [0014]    [0014]FIG. 2. Conidiomata produced on alfalfa stems in distilled water agar.  
         [0015]    [0015]FIG. 3. Conidiomata immersed in leaf of host plant.  
         [0016]    [0016]FIG. 4. Longitudinal section of uniloculate conidiomata produced on host.  
         [0017]    [0017]FIG. 5. Longitudinal section of multiloculate conidiomata produced on PDA.  
         [0018]    FIGS.  6 - 8 . Conidiogenous cells in fluorescence microscopy.  
         [0019]    [0019]FIG. 9. Apical view of conidia showing longitudinal slits in fluorescence microscopy.  
         [0020]    [0020]FIG. 10. Conidia showing basal thickened wall with central pore in fluorescence microscopy.  
         [0021]    [0021]FIG. 11. Median view of conidia.  
         [0022]    [0022]FIG. 12. Surface view of conidia showing widely spaced slits.  
         [0023]    [0023]FIG. 13. Germinating conidia.  
         [0024]    [0024]FIG. 14. Conidia with an appendage. All from holotype (BPI 747560) or ex-type culture. Scale bars: 1-3, 5=100 μm; 4, 9-14=10 μm; 6-8=5 μm.  
         [0025]    FIGS.  15 - 17 .  Harknessia eucalypti . Conidiogenous cells with developing conidia in fluorescence microscopy. Type specimen (BPI 368192) Scale bars in FIGS.  15 - 17 =10 μm.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0026]    Lythrum cultivars were obtained from the Morden Research Station in Morden, Manitoba and independently from Behnke Nurseries in Maryland. These cultivars were propagated in the greenhouse and grown together with other cultivars and weedy lythrum in a loosestrife nursery. One of the inventors observed that some of the cultivars growing in the greenhouse were dying from what appeared to be a plant disease. The sick plants would begin to wilt, and then completely dry out and die. Most of the sick plants were of the cultivar, “Morden Gleam”, which is a hybrid created from crossing the cultivar “Morden Pink” ( Lythrum virgatum ) with the native, winged loosestrife ( L. alatum ). The two species of Lythrum are different species from the weedy purple loosestrife ( L. salicaria ). Although the  L. virgatum  and  L. alatum  are distinct species, they are closely related and will occasionally cross to produce hybrids, as occurred in the creation of the hybrids “Morden Rose” and “Morden Gleam.” Artificial crosses, however, made between the two species have rarely produced seed that will germinate.  
         [0027]    The inventor gave some of the infected plant material to one of the other inventors, who isolated a fungal culture from the cultivars. The inventor then took some of the original fungal culture and sprayed it onto weedy purple loosestrife plants ( Lythrum salicaria ). The inventor was able to reisolate the fungal culture from the weedy purple loosestrife plants. It was also observed that weedy purple loosestrife plants developed the same symptoms as in the “Morden Gleam” plants. Therefore, even though the fungal culture was initially isolated from a hybrid cross of two species,  L. virgatum  and  L. alatum , it was able to infect another closely related, but distinct, species of Lythrum.  
         [0028]    The fungal culture was sent to the U.S. National Fungus Collections for identification. The culture was identified as two fungi,  Coniella frugarei , and a new species,  Harknessia lythrii , which is discussed and illustrated in Example 6 below. The fungal mixture that was isolated from the “Morden Gleam” plant hosts was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 on accession number PTA-223 on Jun. 15, 1999. The newly discovered  Harknessia lythrii  was deposited with the ATCC on Dec. 5, 2000 and accorded accession number PTA-2756 . Coniella frugarei  was deposited with the ATCC on Dec. 5, 2000 and accorded accession number PTA-2757.  
         [0029]    An isolation procedure that may be employed to obtain the fungus or fungal mixture involves surface sterilizing the infected stems or leaves in 70% ethanol for 3-5 minutes, sectioning material with a sterile scalpel, rinsing the material in sterile distilled water, and placing the tissue onto potato dextrose agar. The herbicidal composition can be used in a variety of environments to control growth of noxious weeds, such as purple loosestrife.  
         [0030]    The inventors developed the herbicidal composition of the present invention, which is a mixture formed by combining an effective amount of at least one isolated and purified fungus specific for noxious weeds with a liquid agricultural carrier. The novel mycoherbicide can be used effectively in diverse formulations, including the agronomically acceptable adjuvants and carriers normally employed for facilitating the dispersion of active ingredients for agriculture applications, recognizing a known fact that the dosage, formulations, mode of application of a chemical agent and other variable may affect its activity in any given application. The described mycoherbicide can be formulated as a suspension or dispersion, in aqueous or non-aqueous media, as a dust, as a wettable powder, as an emulsifiable concentrate, as a granule, or as any of several other known types of formulations, depending on the desired mode off application. These herbicide compositions can be applied as sprays, dusts, or granules directly to the plant or its situs where herbicidal activity is desired.  
         [0031]    The subject fungus or fungal mixture can be obtained by conventional culture techniques or from the deposited culture specimens. To convert it to a form that will facilitate the preparation of the following described compositions, a slurry can be prepared that can then be dried onto a primary agronomically acceptable carrier, e.g., vermiculite, whereby the fungus/fungi is adsorbed onto the carrier. If desired, the slurry can be used as the concentrate for the herbicidal composition. The actual concentration of propagules in the formulated composition is not particularly critical, and is a function of practical consideration such as the properties of the vehicle or carrier, and the method of application. Certain spore concentrations, which are described herein, however, have been found to be preferred. For purposes of formulation and application, an “effective amount” is defined to mean any such quantity of propagules sufficient to infect the target plant and thereby induce the lesions involved in the lethal activity described herein.  
         [0032]    The subject material described herein can be applied to a region to be treated by applying it directly to the soil as pre-emergence treatment or as post-emergence treatment to plant foliage, or it can be mixed intimately with the soil. The preferred mode of treatment is application after emergence of the plant foliage. The subject materials described herein can, for example, be applied to soil or plant foliage in amounts of from about 0.1 gallons per acre to 300 gallons per acre, wherein the composition is at a concentration of about 1×10 4  to about 1×10 9  spores per ml.  
         [0033]    As used herein, an “herbicidally effective” amount of the fungal agent is an amount that is sufficient to control the growth of the target plant or plants. “Controlled” plant growth is intended to mean the ability of the fungus according to the present invention to infect its target plant to a degree sufficient to reduce or prevent the ability of the target weed, such as purple loosestrife, to detrimentally affect the growth of the surrounding native or desirable plants. It may kill one or more target plant in the selected area. However, “controlled” growth does not necessarily require the complete eradication of all of the target plants in an area.  
         [0034]    Fungal Agents  
         [0035]    The herbicidal composition of the invention is an effective herbicidal amount of at least one pathogenic fungal agent specific for noxious weeds combined with an agricultural carrier. The noxious weed may be purple loosestrife. The pathogen may be a fungal mixture, in particular, culture deposit ATCC no. PTA-233, or may be an individual fungus, in particular,  Harknessia lythrii , ATCC no. PTA-2756, or  Coniella fragariae , ATCC no. PTA-2757. Suitable fungal agents used alone or in combination are those plant pathogens that are host specific and capable of infecting the host plant and causing disease that interferes with the plant growth and/or reproduction. The plant pathogens are preferably those that selectively infect noxious weeds, such as purple loosestrife, without affecting other plants such as native wetland plants.  
         [0036]    The method of isolating suitable fungal agents involves growing the fungus on potato dextrose agar for 10 days and harvesting spores by scraping the plates in sterile diluted water. Spores are then diluted to 106 spores/ml, as determined by counting in a hemacytometer, and sprayed onto plants.  
         [0037]    Suitable Carriers  
         [0038]    The fungal agent is combined with a suitable carrier in an effective herbicidal concentration. Examples of suitable carriers include water, fertilizers, oils (petroleum or plant based), humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, alginate or clay, including granules or suspensions. Alternatively, the liquid may be modified to yield a physiological solution. Suitable physiological solution include sodium phosphate, sodium chloride, sodium acetate, sodium citrate and the like, preferably in an about 0.001-1M aqueous phosphate buffer. Other suitable physiological solutions are well known in the art and would include 0.85% sodium chloride. An effective herbicidal amount of the fungal agent is preferably 10 2  to 10 12  spores/ml, more preferably 10 4  to 10 9 , and most preferably about 10 5  to 10 8  of the liquid medium.  
         [0039]    Adjuvants and Germination Activators  
         [0040]    An adjuvant may be added to the herbicidal composition of the present invention. The adjuvant provides for relatively low aqueous surface tension and increased plant tissue penetration. The adjuvants are also those that are water-miscible or water-dispersible, easily atomized, capable of being adequately retained on the foliage of plants, have low phytotoxicity, and result in a composition with relatively low surface tension.  
         [0041]    The adjuvants useful in the composition of the present invention are those disclosed in  Adjuvants for Herbicides , Weed Science Society of America, Allen Press, Lawrence, K S, (1982); or  A Guide to Agricultural Spray Adjuvants Used in the United States , Rev. 5th ed., Thompson Publications, (1998), which are incorporated by reference herein. The preferred adjuvants are methylated seed oils. Other types of suitable adjuvants include ampholytic, cationic, anionic, and nonionic.  
         [0042]    The adjuvant is dispersed in the liquid suspension containing the fungal agent, to yield the present herbicidal composition. The adjuvant is present in an effective plant tissue-penetrating amount that is preferably within the range of about 0.001% to 10% volume/volume, more preferably about 0.01% to 6% volume/volume, and most preferably about 0. 1% to 4% volume/volume of the liquid suspension containing the fungal agent.  
         [0043]    The herbicidal composition may also contain a germination activator. Examples would be monosaccharides, disaccharides, polysaccharides, amino acids, peptides, peptones, proteins, inorganic salts, and other solutes.  
         [0044]    Methods of Forming and Using the Herbicidal Composition  
         [0045]    The herbicidal composition is useful to control weed growth in a variety of environments, especially wetlands. These environments generally have a mixed population of plants, for example, cattail (Typha spp.), beggartick (Bidens spp.), broad-leaved arrowhead ( Sagittaria latifolia ), bulrush (Scirpus spp.) and willow (Salix spp.). The composition is effective against purple loosestrife without affecting the other wetland plants. The herbicidal composition is also suitable for application by low pressure spraying so that large areas of land may be easily treated.  
         [0046]    The present herbicidal composition is formed by combining an effective amount of at least one fungal agent with an agricultural carrier, and optionally with an adjuvant and/or a germination activator, to form an essentially homogeneous dispersion.  
         [0047]    After the herbicide is formulated, it is applied to the weed-infested area. The weed population includes purple loosestrife. Other wetland plants are not inhibited. The herbicidal mixture may be applied by ground spraying, aerial spraying, painting or brushing, or by hand or mechanical dispersion, including but not limited to backpack or other hand held devices, hydraulic or air nozzles, granular applicators, electrostatic applicators, controlled droplet applicators (CDA), or ultra-low volume (ULV) applicators. The herbicidal composition of the present invention is especially suitable for spraying.  
         [0048]    The herbicidal mixture is applied in single or repeated applications until weed growth is effectively inhibited. The conditions leading to effective weed growth inhibition depend, in part, on the environment. For example, a single application of the herbicidal mixture may be sufficient or a plurality of application may be required. The herbicidal composition of the present invention can be applied to bare ground, plant litter or to plants of any age to inhibit growth and/or reproduction, including plants that have flowered or senesced. The herbicidal mixture is applied at a density sufficient to cover the area where weed growth is expected to be observed in amounts from about 0.1 gallons per acre to 300 gallons per acre, wherein the composition is at a concentration of about 1×10 2  to about 1×10 12  spores per ml. The concentration may be in the range of 10 9  to 10 9  spores per ml, or in the range of 10 5  to 10 8  spores per ml. Weed growth is effectively inhibited if the majority of weeds are infected with the fungal agent and exhibit symptoms of disease.  
         [0049]    The following examples are intended to illustrate but not limit the invention.  
       EXAMPLE 1  
     Treatment of Purple Loosestrife with a Combination of a Fungal Agent and  Galerucella calmariensis    
       [0050]    The fungal agent deposited as ATCC PTA-223 produced in culture was applied in distilled water with a hand-pump sprayer. The spray solution was applied at a concentration of 1.3×10 6  spores/ml to weedy purple loosestrife plants in combination with the leaf defoliating beetle,  Galerucella calmariensis . Adult beetles were placed in an un-capped vial. Vials were placed inside screen cages covering the plants. Beetles crawled out of vials and onto plants.  G. calmariensis  is an insect biological control agent currently being released in Minnesota to control purple loosestrife. Leaf feeding by the beetle may provide entry wounds for the fungal agent. After a period of eight days, the insects were removed by hand from purple loosestrife plants and the plants were evaluated for fungal disease symptoms. Stem cankers were found on plants. Ten plants were tested, and all plants with the fungal agent +dH 2 O had cankers and the cankers were determined to be caused by the fungal agent. On half of the plants there was no green tissue remaining.  
       EXAMPLE 2  
     Field Treatment of Purple Loosestrife with a Fungal Agent  
       [0051]    In another experiment, the inventors sprayed purple loosestrife with fungal agent ATCC PTA-223 in a wetland at the Dodge Nature Center in Mendota Heights, Minn. in late July. The fungal agent was sprayed as a liquid formulation with a backpack sprayer at a concentration of 1×10 6  spores/ml in combination with 3% AGRI-DEX® which is manufactured by Helena Chemical Co., Memphis, Tenn. In one treatment, spores were obtained from plate culture and in the other treatment, spores were obtained from shake culture. Plates were prepared for potato dextrose agar and broth was prepared for potato dextrose broth. The cultures were grown at room temperature for 10 days, and shake cultures were shaken on an orbital shaker at 250 RPM. Two, 10 by 10 feet plots were sprayed for each treatment. Plants were evaluated for presence of the fungal agent in late August of that same year. The fungal agent was reisolated from purple loosestrife plants from the plate culture treatment.  
       EXAMPLE 3  
     Greenhouse Treatment of Purple Loosestrife with a Fungal Agent  
       [0052]    A greenhouse study was conducted in which purple loosestrife seedlings were sprayed with fungal agent ATCC PTA-223 in combination with different spray additives. Emery 6804 which is manufactured by Henkel, Cincinnati, Ohio, AGRI-DEX® and SOYDEX® which is manufactured by Helena Chemical Co., Memphis, Tenn. were used at 3% v/v. Spore concentration was 1.0×10 6  spores/ml. Fungal stem cankers developed on purple loosestrife stems on all treated plants. The greatest disease severity and incidence occurred on plants treated with Emery 6804. The controls (not sprayed) did not become infected.  
       EXAMPLE 4  
     Treatment of Potted Purple Loosestrife with the Fungal Mixture  
       [0053]    In the spring, 60 perennial purple loosestrife crowns were planted in pots and allowed to grow. When shoots were from 4 to 8 inches tall, they were treated with one of the following: fungal mixture ATCC PTA-223 plus Emery 6804 (3% v/v); control plus Emery 6804 (3% v/v); and control (no treatment). Plants were rated for disease incidence and percent tissue necrosis after one month. At this time, half of the plants were cut and aboveground biomass measurements were obtained. These plants were then resprayed when the shoots were 4 to 8 inches in height for a total of two treatments per plant. After one month, data was collected as described above. Plants sprayed one time only were mulched and overwintered. In the following summer, aboveground biomass was determined for the overwintered plants (see Table 1).  
                                           TABLE 1                           Efficacy of the fungal mixture on purple loosestrife regrowth dry       weights one year after application.                    Dry Weight           Treatment   -g-                            Control   50.0           Control + Emery 6804   50.7           Control + Emery 6804   62.1           (2 applications)           Fungal Mixture +   8.9           Emery 6804           Fungal Mixture +   23.1           Emery 6804           (2 applications)           LSD (0.05)   30.5                      
 
         [0054]    Results of this experiment indicated that plants treated with the fungal mixture showed disease symptoms and the fungus was reisolated from disease lesions. Control and control plus adjuvant treatments did not have disease symptoms. Crown regrowth, from plants treated two times in the first summer, was reduced as compared to control plants. Plants overwintered and harvested in the following summer also had statistically less significant regrowth as compared to control treatments. This was a significant result as it showed that crown growth may be impaired after treatment with the fungal mixture, an important consideration when trying to control a persistent perennial plant such as purple loosestrife. Control of regrowth from the crown is critical for long-term suppression of purple loosestrife.  
       EXAMPLE 5  
     Specificity of the Fungal Mixture as a Biological Control Agent  
       [0055]    This experiment was conducted in wetlands located at Roseville Central Park, Roseville, Minn. In July, plots were sprayed with Emery 6804 at 3% v/v, and two rates of spores of the fungal mixture ATCC PTA-223; 1×10 6  spores/ml or 2×10 6  spores/ml. At the time of spraying plants were approximately 3 to 5 feet tall and were flowering. After one month, purple loosestrife plants were rated for disease incidence. Established purple loosestrife plants had noticeable disease lesions, which when cultured, were identified as the fungal mixture. Other wetland plants, such as cattail (Typha spp.), beggartick (Bidens spp.), broad-leaved arrowhead ( Sagittaria latifolia ), bulrush (Scirpus spp.) and willow (Salix spp.) that were sprayed with the mycoherbicide did not exhibit similar lesions and the fungal mixture could not be isolated from these species. Seedling purple loosestrife plants were killed by treatments with the fungal mixture.  
         [0056]    In the following summer, the plots sprayed the previous summer were evaluated. Fungal lesions were found on stems of plants treated in the previous year, and on stems of new growth. When the lesions were examined, characteristic pycnidia were present containing spores of the fungal mixture. This experiment showed that the fungal mixture survived the winter and was able to re-infect purple loosestrife plants the following summer. This is an important attribute for a mycoherbicide used for control of purple loosestrife, as herbicide application costs are very expensive due to site inaccessibility.  
       EXAMPLE 6  
     Identification of  Harknessia lythrii  on Purple Loosestrife  
       [0057]    Material and Methods  
         [0058]    For microscopic examination material was rehydrated and mounted in 3% KOH. Conidiomata were sectioned at about 10 μm thick using a freezing microtome. Sections were mounted in lactic acid with cotton blue. Observations of microscopic features were made using a Zeiss Axioplan 2 microscope with both bright-field and fluorescence illumination. Calcofluor was used as the fluorescent dye. Photographs and measurements of microscopic features were taken using a Spot 2 digital camera (Diagnostic Instruments, Inc., Sterling Heights, Mixh.) and ImagePro software (Media Cybernetics, Silver Spring, Md.). The fungus was maintained on Difco Corn Meal Agar (CM) slants with an alfalfa stem at 4° C. and in water cultures (Burdsall, Jr. H. H., Dorworth, E. B., Preserving cultures of wood-decaying Basidiomycotina using sterile distilled water in cryovials.  Mycologia,  86, 275-280 (1994)). To produce and observe pycnidia in culture the fungus were grown on autoclaved 20-40×1-2 mm stems of Medicago (alfalfa) placed on distilled water agar in petri dishes. For the growth studies, plates of CM and PDA were inoculated with a 6 mm diam plug of actively growing mycelium placed at the edge of the plate and grown at 25° C. in the dark for eight days. Color names and numbers were determined using Kornerup &amp; Wanscher (1978),  Methuen Handbook of Colour  London: Methuen &amp; Co. (243 pp.).  
         [0059]    Taxonomy  
         [0060]    [0060] Harknessia lythrii  Farr &amp; Rossman, sp. nov. FIGS.  1 - 14  Conidiomata in hospite uniloculata, globosa 75-90 μm, bruneola, per rimam elongatam dehiscentia; cellulae conidiogenae basicis lateralibusque, determinatae vel uno or duo prolieratione, percurrent, annularibus; conidia brunnea ad atro-brunnea, subglobosa ad irregulariter ellipsoidea, 10.6-18.5×8.9-15.4 μm, unicellulosa, 2-7 aequaliter dispositis, angustis, longitudinalibus striis, basi breviter eminentia, subinde brevi appendice. Holotype: UNITED STATES: Minnesota: St. Paul, on  Lythrum salicaria  in greenhouse. BPI 747560.  
         [0061]    Conidiomata often obscured by extruded dark-brown conidia; on host conidiomata immersed to semi-immersed, subcuticular, scattered, stromatic, uniloculate, globose, 75-90 μm diam, glabrous, opening by an elongated slit, wall thin, of 2-3 layers of textura angularis, outer two layers pale brown, outer layer of cells with somewhat thickened walls; on alfalfa stems, conidiomata similar to those on host except slightly larger, 90-150 μm diam, wall of 3-5 layers of textura angularis, outer two layers often of thick-walled cells; on PDA, conidiomata superficial, appearing black due to dark-brown due to conidia appearing through pallid wall, covered with loosely intertwined, hyaline mycelium, scattered to clustered, stromatic, multiloculate, globose to slightly elongated, up to 1200 μm diam, each locule opening by an elongated slit, locular wall of 3-5 layers of textura angularis, outer layer of pale brown cells with slightly thickened walls. Conidiophores lacking. Conidiogenous cells lining the base and sides of the conidiomata, ampulliform, 6-10×3.5-6.1 μm, with a short to elongated apex bearing conidia, once or twice annellidic following percurrent proliferation of apex, length of proliferation variable, abscission layer between apex of conidiogenous cell and base of developing conidia evident in fluorescent microscopy, base of conidia thickening centripetally with a cytoplasmic connection remaining as a central pore, evident in fluorescent microscopy. Conidia brown to dark brown, subglobose to irregularly ellipsoidal, 10.6-18.5 μm ({overscore (x)}=14.7, SD=1.5, n=156)×8.9-15.4 μm ({overscore (x)}=11.6, SD=1.25), unicellular, wall up to 1.5 μm thick, with 2 to 7 widely spaced, narrow, longitudinal slits extending from the apex to the base, base truncate, with a short flange, occasionally with a short basal appendage. Microconidia not seen. On PDA colony 70 cm diam, white to light yellow (4A5), zonate, zones about 2, 0.7, 3.5 cm from plug, surface mycelium cottony, margin feathery, reverse pale yellow (4A3); on CM colony 61 cm diam, translucent, no zones, scattered cottony strands of surface mycelium, margin even.  
         [0062]    Type specimen: UNITED STATES: Minnesota: St. Paul, on  Lythrum salicaria  in greenhouse. (HOLOTYPE-BPI 747560; ex-type culture deposited as ATCC no. PTA-2756). This species and a second fungus,  Coniella fragariae  (Oud.) B. C. Sutton (ATCC no. PTA-2757), were isolated from a greenhouse-grown plant of purple loosestrife cultivar ‘Morden gleam’ which is a hybrid of  Lythrum virgatum  L. and  L. alatum  Pursh. The cultured isolate of  H. lythri  was sprayed on  Lythrum salicaria  in the greenhouse and produced disease symptoms as well as sporulating on the leaves. The holotype specimen was made from the artificially inoculated plant.  
         [0063]    Results  
         [0064]    [0064] Harknessia lythri  is characterized by conidiomata that are stromatic locules each opening by an elongated slit (FIGS.  1 -5). On the host and on alfalfa stems in culture, conidiomata of  H. lythri  are small, up to 150 μm diam, and uniloculate (FIGS.  2 - 4 ) but become quite large up to 1200 μm diam and multiloculate on agar alone (FIGS. 1, 5). In  H. lythri  and  H. eucalypti , the type of Harknessia, conidiophores are lacking and the conidiogenous cells line the base and sides of the locule (FIGS.  4 - 5 ). In  H. eucalypti  there are two distinct layers between the conidia attached to the conidiogenous cell that are evident using fluorescent microscopy (FIGS.  15 - 17 ). One layer is between the conidial body and the conidial appendage and another between the apex of the conidiogenous cell and what will become the base of the appendage. As the conidia mature, the abscission layer closest to the conidiogenous cell becomes more distinct and eventually becomes the point of detachment (FIG. 16). This point of detachment determines the length of the conidial appendage characteristics of many species of Harknessia. This second abscission layer is not evident in  H. lythri  although the conidial development is similar (FIGS.  6 - 8 ). As the holoblastic conidia of  H. lythri  develop, the base of the conidium thickens centripetally with a cytoplasmic connection remaining as a central pore (FIG. 10). The conidia of  H. lythri  are unicellular and dark-brown (FIGS.  9 - 14 ) and have multiple, widely spaced, narrow longitudinal slits on the conidia that extend from the apex to the base (FIGS. 9, 12). Although multiple slits are present, only one tube is formed upon conidial germination (FIG. 13). In  H. lythri  the basal appendage or frill, typical of species of Harknessia, may be reduced or almost non-existent although conidia with an appendage are occasionally seen (FIG. 14).  
         [0065]    Discussion  
         [0066]    Harknessia is a coelomycetous genus that has been well characterized by Sutton, B. C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980); and Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993). The characteristics of Harknessia include having immersed, uniloculate, pale brown conidiomata of thin-walled, pale brown to hyaline, small-celled textura angularis (Sutton, B. C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980)). The conidiomata of  Harknessia lythri  on the host and on alfalfa stems in culture are uniloculate (FIGS.  2 - 4 ), however, when produced on agar alone, they are much larger and multiloculate (FIGS. 1, 5), suggesting that the conidiomatal size is variable and that the distinction between uniloculate and multiloculate conidiomata is not a major one. The increase in the size and wall thickness of coelomycetous fruiting bodies when produced on agar substrate has been noted previously by Farr, D. F., Bills, G. F.,  Wojnowicia colluvium  sp. nov. isolated from conifer litter.  Mycologia,  87, 518-524 (1995) who emphasize the importance of producing fruiting bodies on plant material in culture in order to simulate natural conditions. Although Sutton, B. C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980) describes the conidiomata of Harknessia as having an “ostiole central, circular, wide, unspecialized, furfuraceous,” Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993) states that the conidiomata are “lacking an ostiole but dehiscing by circular or irregular breaks in the apical covering layer” as was observed in  H. lythri.    
         [0067]    Conidiogenesis cells in Harknessia are described as lining the base and sides of the conidiomata as occurs in H. lythri (FIGS. 4, 5). This feature distinguishes Harknessia from Coniella Höhn., a coelomycetous genus having conidia similar to those of Harknessia. In Coniella, the conidiogenesis cells arise from a cushion of tissue at the base of the conidiomata (Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993); Sutton, B.C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980)). In Harknessia conidiophores are generally lacking. The conidiogenous cells are determinate, often ampulliform, lageniform, subcylindrical or cylindrical, producing conidia holoblastically, often proliferating percurrently to produce additional conidia at the same or higher level (Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993)). The conidiogenous cells of  H. lythri  are ampulliform and often extend slightly upon the production of the second or third conidium (FIGS.  6 -8). Species of Harknessia have smooth, brown, unicellular conidia, sometimes longitudinally striate, “with a cellular unbranched basal appendage resulting from the persistent conidiogenous cells” (Sutton, B. C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980)). The smooth, dark brown, unicellular conidia of  H. lythri  are characteristic of Harknessia, although the conidial ornamentation and lack of a distinct appendage are unusual as discussed below, distinguishes  H. lythri  from all other species of Harknessia.  
         [0068]    The most comprehensive account of the genus Harknessia is presented by Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993) who described and illustrated 26 species. Ten additional species have been included in the genus, specifically  H. ventricosa  Sutton &amp; Hodges on Eucalyptus (Sutton, B.C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980));  H. karwarrae  Sutton &amp; Pascoe on  Acacia glucoptera  (Fabaceae) and  H. victoriae  Sutton &amp; Pascoe on Eucalyptus (Sutton, B. C., Pascoe, I., Addenda to Harknessia (Coelomycetes). Mycol. Res, 92, 431-439 (1989));  H. eucalyptorum  Crous et al. and  H. fusiformis  Crous et al., both on Eucalyptus, and  H. syzygii  Crous et al. on  Syzygium coradum  (Myrtaceae) (Crous, P. W., Wingfield, M. J., Nag Raj, T. R., Harknessia species occurring in South Africa.  Mycologia,  85, 108-118 (1993));  H. leucospermi  Crous &amp; L. Viljoen on Leucospermum (Proteaceae) (Swart, L., Crous, P. W., Denman, S., Palm, M. E., Fungi occurring on Proteaceae.  I. South African J. Bot.,  64, 137-145 (1998)); H. salvertiana Furlan. &amp; Dianese on  Salvertia convallarioidora  and  H. qualeae  Furlan. &amp; Dianese on  Qualea grandiflora  (both hosts in the Vochysiaceae) (Furlanetto, C., Dianese, J. C., Some coelomycetes from Central Brazil.  Mycol. Res.,  102, 19-29 (1998)); and  H. tasmaniensis  Z. Q. Yuan et al. on Eucalyptus (Yuan, Z. Q., Wardlaw T., Mohammed, C., Harknessia species occurring on eucalypt leaves in Tasmania, Australia. Mycol. Res., 104, 888-892 (2000)). A few additional names exist in Harknessia but cannot be adequately characterized (Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993)).  
         [0069]    Species of Harknessia are variable in their conidial ornamentation ranging from completely smooth to having narrow or broad, single or multiple, closely spaced or widely spaced longitudinal striations. Most species of Harknessia have one longitudinal striation evident as a pallid band of varying widths or have 2-5 closely spaced, narrow longitudinal striations evident as a band on only one side of the conidium. Whether these striations are actually slits has not been determined except in the case of  H. lythri  in which germination occurs through this structure (FIG. 13). One species of Harknessia with unusual conidial ornamentation is  H. gharsei  Golatkar in which conidia have widely spaced, broad, longitudinal bands. A few species of Harknessia have conidia that lack any striations such as in  H. americana  (Mont.) Sutton,  H. caudata  Ellis &amp; Everh.,  H. deightonii  Sutton,  H. eucrypta  (Cooke &amp; Massee) Nag Raj &amp; DiCosmo,  H. fusca  (Klebahn) Nag Raj &amp; DiCosmo, H. insueta Sutton, H. liquidambaris (Berk. &amp; Curt) Nag Raj &amp; DiCosmo and  H. shearii  Petrak. Thus, the widely spaced, narrow, longitudinal slits of  H. lythri  are unusual and distinct but fall within the range of conidial ornamentation that occurs among species of Harknessia.  
         [0070]    In  Harknessia lythri  the basal appendage on the conidia is reduced to a basal frill or appendage seen only occasionally (FIG. 14). For several species included in Harknessia such a conidial appendage is also very short or lacking. These species include  H. guunerae  Stevens &amp; Young, in which the appendage is described as 2-3 μm long, and  H. insueta  in which the appendage is described as 1.5 μm long but not illustrated in Sutton, B. C., The Coelomycetes. Kew, Surrey: Commonwealth Mycological Institute, p. 696 (1980). Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993) stated that the appendage of  H. insueta  may be up to 2 μm long but is “often gelatinizing and resulting in a minute marginal frill on the truncate base of the conidium”. Thus, the lack of a basal appendage does not exclude  H. lythri  from the genus Harknessia.  
         [0071]    The species of Harknessia most similar to  H. lythri  are  H. gharsei, H. globosa  Sutton and  H. hawaiiensis  Stevens &amp; Young. All of these four species have subglobose to broadly ellipsoid conidia that are about the same size.  Harknessia gharsei  has conidia with widely spaced “longitudinal bands of brown and yellowish-brown” (Nag Raj, T. R., Coelomycetous Anamorphs with Appendage-bearing Conidia. Waterloo, Ontario: Mycologue Publications, p. 1101 (1993)), rather than narrow slits as in  H. lythri. Harknessia gharsei  is similar to  H. lythri  in having conidia each with a very short or non-existent basal appendage, however, in addition to the differing conidial ornamentation, the conidia of  H. gharsei  also have a distinct apiculus. Both  H. globosa  and  H. hawaiiensis  have conidia with narrow longitudinal striations in a restricted area on only one side of the conidium, and distinct basal appendages 3-10 μm long.  
         [0072]    Most species of Harknessia occur on the plant host Eucalyptus (Myrtaceae); other members of the Myrtaceae such as Melaleuca, Metrosideros, and Syzygium; plant families in the Myrtales such as the Lythraceae, Melastomataceae and Vochysiaceae; and plants with leathery or evergreen leaves such as the Araucariaceae, Ericaceae, Podocarpaceae, Proteaceae, and Smilaceae. The host for  H. lythri, Lythrum salicaria , belongs in the Lythraceae, Myrtales, and is considered to have affinities with the Myrtaceae (Heywood, V. H., ed., Flowering Plants of the World. New York, New York: Mayflower Books, p. 335 (1978)). Most species of Harknessia are known from only one host plant genus. The exceptions include  H. uromycoides  (Speg.) Speg., originally described on Eucalyptus, now also known from  Platylobium obusangulum  (Fabaceae) and  Banksia marginata  (Proteaceae);  H. renispora  Swart described on Melaleuca (Myrtaceae), now reported on other myrtaceous hosts as well as a non-myrtaceous host,  Alyxia buxifolia  R. Br. (Apocynaceae); and  H. thujina  Ellis &amp; Everh. known only from members of the Cupressaceae. At present,  H. lythri  is known only from  Lythrum salicaria . In general members of the genus Harknessia are known on tropical and semi-tropical plants with exceptions. The type species,  H. eucalypti , was originally described from the San Francisco area of California;  Harknessia americana  and  H. fuegiana  Speg. are reported from Chile;  H. antarctica  Speg. is described from Argentina;  H. liquidambaris  is reported from New Jersey and Georgia;  H. sudans  (Petrak) Nag Raj occurs in France; and  H. thujina  is known from New Jersey, Texas and Canada. Although  H. lythri  is reported here from Minnesota, the family Lythraceae is described as having a mainly tropical, sometimes temperate, distribution (Heywood, V. H., ed., Flowering Plants of the World. New York, New York: Mayflower Books, p. 335 (1978)).  
         [0073]    All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the scope of the invention.