The Effect of Temperature on Disease Severity and Growth of Fusarium oxysporum f. sp. apii Races 2 and 4 in Celery.

Fusarium oxysporum f. sp. apii race 4, which is in F. oxysporum species complex (FOSC) Clade 2, causes a new Fusarium wilt of celery. We compared F. oxysporum f. sp. apii race 4 with race 2, which causes Fusarium yellows of celery and is in FOSC Clade 3. Optimal temperatures for celery yield are 16 to 18°C. Soil temperatures in California celery production areas can range up to 26°C, and the maximal rate of hyphal extension of F. oxysporum f. sp. apii races 2 and 4 in culture are 25 and 28°C, respectively. Here, we compared the effect of temperatures from 16 to 26°C on growth of F. oxysporum f. sp. apii races 4 and 2 in two celery cultivars: Challenger, which is resistant to F. oxysporum f. sp. apii race 2 and susceptible to race 4; and Sonora, which is susceptible to both F. oxysporum f. sp. apii races 2 and 4. Based on linear regressions, as temperature increases, there is an increase in the log of F. oxysporum f. sp. apii race 4 DNA concentration in celery crowns and in the reduction in plant height. Based on logistic regressions, as temperature increases, the incidence of vascular discoloration increases in celery with either F. oxysporum f. sp. apii race 2 or 4 infection. In both cultivars, temperatures of 22°C and above resulted in a significantly (α = 0.05) greater concentration of F. oxysporum f. sp. apii race 4 than race 2 in planta. The concentration of F. oxysporum f. sp. apii race 2 in crowns in 'Challenger' is temperature-independent and comparatively low; consequently, 'Challenger' is, at least partly, resistant rather than tolerant to F. oxysporum f. sp. apii race 2.

Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii.

BACKGROUND: Members of the F. oxysporium species complex (FOSC) in the f. sp. apii (Foa) are pathogenic on celery and those in f. sp. coriandrii (Foci) are pathogenic on coriander (=cilantro). Foci was first reported in California in 2005; a new and highly aggressive race 4 of Foa was observed in 2013 in California. Preliminary evidence indicated that Foa can also cause disease on coriander, albeit are less virulent than Foci. Comparative genomics was used to investigate the evolutionary relationships between Foa race 4, Foa race 3, and the Foci, which are all in FOSC Clade 2, and Foa race 2, which is in FOSC Clade 3. RESULTS: A phylogenetic analysis of 2718 single-copy conserved genes and mitochondrial DNA sequence indicated that Foa races 3 and 4 and the Foci are monophyletic within FOSC Clade 2; these strains also are in a single somatic compatibility group. However, in the accessory genomes, the Foci versus Foa races 3 and 4 differ in multiple contigs. Based on significantly increased expression of Foa race 4 genes in planta vs. in vitro, we identified 23 putative effectors and 13 possible pathogenicity factors. PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified. Finally, mixtures of conidia that were pre-stained with different fluorochromes indicated that Foa race 4 formed conidial anastomosis tubes (CATs) with Foci. Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other. CONCLUSIONS: There was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4; Foa race 2 and 4 are CAT-incompatible. Although Foa races 3 and 4 and the Foci are closely related, there is no evidence that either Foci contributed to the evolution of Foa race 4, or that Foa race 4 was the recent recipient of a multi-gene chromosomal segment from another strain. However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range.

The small GTPase BcCdc42 affects nuclear division, germination and virulence of the gray mold fungus Botrytis cinerea.

The small GTPase Cdc42 plays a central role in various processes in eukaryotic cells including growth, differentiation and cytoskeleton organization. Whereas it is essential in the yeast Saccharomyces cerevisiae, its role in filamentous fungi differs, due to the complementing, partly overlapping function of Rac. We analyzed the role of the Cdc42 homologue in the necrotrophic, broad host range pathogen Botrytis cinerea. Deletion mutants of bccdc42 showed various growth abnormalities; the mutants had reduced growth rate and hyphal branching, they produced fewer conidia, which were enlarged and misshapen and had germination defects. Additionally, the mutants were impaired in sclerotia development. Cytological studies indicate that at least part of this phenotype could be attributed to disturbed control of nuclear division: conidia and hyphae of the mutant showed twofold higher nucleus/cytoplasm ratio compared to wild type cells. Apart from these effects on vegetative growth and differentiation, Δbccdc42 strains were attenuated in penetration and colonization of host tissue, confirming that BcCdc42 - though being not essential like in yeast - is involved in important developmental processes in B. cinerea.

The cAMP-dependent signaling pathway and its role in conidial germination, growth, and virulence of the gray mold Botrytis cinerea.

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as alpha subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Deltabcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Deltabac, lesion development of Deltabcpka1 and DeltabcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Deltabac, Deltabcpka1 and DeltabcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Deltabcpka1 and DeltabcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Galpha subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.

Ethylene biosynthesis in Botrytis cinerea.

Ethylene is often released during plant pathogenesis. Enhanced ethylene biosynthesis by the attacked plant, and formation of ethylene by the attacking pathogen may be involved. We defined the biosynthetic pathway of ethylene in the pathogenic fungus Botrytis cinerea, and characterized the conditions that affect ethylene production in vitro. During the first 48 h of culture the fungus uses methionine to produce alpha-keto gamma-methylthiobutyric acid (KMBA) and secretes it to the medium. In darkness, KMBA accumulates in the medium. In light KMBA is photo-oxidized and ethylene is released. The photo-oxidation reaction is spontaneous and does not involve any enzymatic activity. Low levels of ethylene are produced in darkness between 48 and 96 h of culture. Adding peroxidase to dark-grown cultures induced ethylene formation. The results suggest that formation and secretion of KMBA by B. cinerea may affect ethylene levels during plant infection.