Pathogen Biology

Monosporascus cannonballus is a pyrenomycete (Ascomycota: Sordariales) fungus, although the exact taxonomic placement probably needs re-examination. It forms large (40-50 µm-diam), spherical, multi-layer walled ascospores (Figures 6 & 7), however typically only one ascospore is formed per ascus instead of the usual eight (Figure 8). Ascospores are multinucleate, typically having eight nuclei, although occasionally they may have 16. There is no known anamorph (asexual) stage. Four species of Monosporascus have been described, but only two, M. cannonballus and M. eutypoides, have been implicated in disease of cucurbits. Both are pathogenic to melons and they were originally distinguished from each other on the basis of the number of ascospores per ascus and whether or not they germinated readily. Studies based on partial DNA sequence similarity, pathogenicity and other phenotypic traits, suggested that they are conspecific (the same species). More recent data on how the ascospores germinate and the number of germ tubes formed argue for maintaining them as separate species. However, isolates of M. eutypoides are difficult to locate making direct comparisons difficult. Since M. cannonballus is the original species name (holotype) and it is the major pathogen of vine decline reported around the world, its name has been retained in this paper.

Figure 6.	Figure 7.	Figure 8.

The fungus is homothallic (self-fertile) and readily forms fertile perithecia in host root tissue (Figures 3 and 4) and in vitro on artificial growth media (Figure 5). Ascospores are thick, multi-walled spores (Figures 6, 7, and 8) and are extremely resistant to desiccation and other factors. Germination of the ascospore is rare in vitro, however germination is enhanced in situ when in the presence of root exudates from growing muskmelon seedlings. Soil microflora, most likely actinomycetes also are important in the germination of ascospores in the field. Monosporascus cannonballus is adapted to hot, dry climates. In vitro vegetative growth is optimal at 25 to 35°C (77 to 95°F), while perithecia are formed most readily at 25 to 30°C (77 to 86°F). The fungus may survive for several days at temperatures up to 55 C°, but is killed within 90 min at 60°C. Mycelial growth occurs over a pH range of 5 to 9, but is optimal from pH 6 to 7 and inhibited completely at pH 4 and below. Monosporascus cannonballus appears also to be adapted to slightly or moderately alkaline and saline soils. The fungus grows readily on several standard laboratory growth media (e.g. potato dextrose agar, V-8 juice agar, and water agar) and forms fertile black perithecia within 2 to 3 weeks. Perithecia are readily visible against the light gray or dirty white mycelium.

In culture (e.g., PDA, V-8 juice agar, water agar), wild-type Monosporascus colonies typically are a dirty-white or light grey color with numerous black perithecia embedded in the agar. Under some circumstances, colonies may produce pigments giving the mycelia a yellow to orange to dark brown color. Pigmented colonies are slow-growing, produce few, if any, perithecia, and are often hypovirulent. Colony death often occurs in heavily pigmented cultures. Five different hexaketides have been identified from pigmented cultures. Little is known as to why some colonies develop these dark pigments but dsRNA may play a role. dsRNA also may play a role in decreased perithecia production and hypovirulence.

There appears to be little genetic variation within M. cannonballus. Although a detailed analysis of a large population has not been done, selected isolates of M. cannonballus from several countries, including the U. S., Japan, and Spain, were identical in colony morphology, number of ascospores/ascus (one), virulence to melon, and DNA sequence across the internal transcribed spacer regions (ITS 1 and 2) and 5.8S gene of the rDNA, suggesting that most isolates are genetically very similar. There do appear to be some differences in vegetative compatibility among U.S. isolates and those from other regions of the world. This aspect has not been studied in depth and could add valuable information to understanding the genetics of this fungus.

In some instances, extreme variations in colony appearance, including the formation of dark yellow, orange or brown pigments (Figure 14), reduced vegetative growth, and loss of perithecia production, as well as loss of virulence and colony death, have been reported. Isolates displaying one or more of these phenotypes harbored one or more double-stranded RNAs (dsRNA), which were attributed to causing the aberrant phenotype. Several distinct dsRNA groupings have been identified. This has been explored as a potential biocontrol of MRR/VD.

Figure 14.

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by The American Phytopathological Society