Survival, Host-Pathogen Interaction, and Management of Macrophomina phaseolina on Strawberry in Israel.

Crown and root rot of strawberry, caused by Macrophomina phaseolina, have become predominant soilborne diseases of strawberry in Israel over the past 5 years. In total, 151 isolates of the pathogen were isolated from infected strawberry plants of commercially grown cultivars in Israel onto a modified agar medium for the genus Macrophomina. Sclerotia viability declined more rapidly in soil maintained at 25°C or at soil temperatures fluctuating from 18 to 32°C under greenhouse conditions, compared with sclerotia viability in soil kept at 30°C. After 30 to 40 weeks of exposure in soil, inocula maintained at 25 or 30°C or at fluctuating temperatures in a greenhouse declined to negligible levels. A significant increase in plant mortality was observed in infested soils maintained at 30 versus 25°C, whereas water stress at 25 or 30°C did not affect plant mortality in M. phaseolina-infested soils. This demonstrated the importance of elevated soil temperature, not moisture stress, on plant mortality caused by M. phaseolina. Host specificity was not evident when strawberry plants were inoculated with each of seven Israeli isolates of M. phaseolina obtained from six other plant species, suggesting the importance of keeping strawberry crops out of rotation with other host crops of the pathogen. The soil fumigants methyl bromide (applied at 500 kg/ha) and metam sodium (730 liter/ha) caused 90 and 95% pathogen mortality in field experiments, respectively, indicating that fumigation may be an effective method of managing this pathogen in infested soils. The increase in prevalence of crown and root rot caused by M. phaseolina in strawberry crops in Israel may be related to the phase-out of methyl bromide.

Identification and Characterization of Benomyl-Resistant and -Sensitive Populations of Colletotrichum gloeosporioides from Statice (Limonium spp.).

ABSTRACT Sixty-four isolates of Colletotrichum gloeosporioides were isolated from infected Limonium spp. cultivated in 12 different locations in Israel. All isolates were identified as belonging to the C. gloeosporioides complex by species-specific primers. Of these isolates, 46 were resistant to benomyl at 10 mug/ml and 18 were sensitive to this concentration of fungicide. Based on arbitrarily primed polymerase chain reaction of all isolates and internal transcribed spacer-1 sequence analyses of 12 selected isolates, the benomyl-resistant and -sensitive populations belong to two distinct genotypes. Sequence analyses of the beta-tubulin genes, TUB1 and TUB2, of five sensitive and five resistant representative isolates of C. gloeosporioides from Limonium spp. revealed that the benomyl-resistant isolates had an alanine substitute instead of a glutamic acid at position 198 in TUB2. All data suggest that the resistant and sensitive genotypes are two independent and separate populations. Because all Limonium plant propagation material is imported from various geographic regions worldwide, and benomyl is not applied to this crop or for the control of Colletotrichum spp. in Israel, it is presumed that plants are bearing quiescent infections from the points of origin prior to arrival.

Isolation of Nonpathogenic Mutants of Fusarium oxysporum f. sp. melonis for Biological Control of Fusarium Wilt in Cucurbits.

ABSTRACT Two nonpathogenic mutant strains 4/4 and 15/15 of Fusarium oxysporum f. sp. melonis (race 1,2) were isolated by a continuous dipinoculation technique following UV mutagenesis of the virulent wild-type isolate FOM1.2. No disease symptoms or detrimental effects were observed following inoculation of muskmelon seedlings by strain 4/4. In contrast, strain 15/15 caused mortality of susceptible cultivars although to a lesser extent than the wild-type isolate. Strain 4/4 colonized a variety of muskmelon and watermelon cultivars. In muskmelon cv. Ein Dor, seedlings were dipped in a conidial suspension of strain 4/4 and planted in medium amended with the mutant to achieve 100% colonization of roots and between 30 to 70% of the lower stem tissues 7 days after planting. Similar percent colonization of watermelon seedlings by strain 4/4 was recorded. In cross-protection experiments with muskmelon cultivars, significant reduction in seedling mortality was observed between 4/4-colonized FOM1.2. challenged plants compared with that of wild-type challenged plants alone. Similarly, strain 4/4 was able to significantly reduce mortality of watermelon seedlings caused by F. oxysporum f. sp. niveum race 2. This novel approach of generating nonpathogenic mutants for biological control in Fusarium spp. and other fungal pathogens from virulent wild-type isolates may be beneficial for control, because the mutant strains, lacking only in pathogenicity, may compete more efficiently than other biocontrol organisms against the pathogen of origin.

Development of a Robust Screening Method for Pathogenicity of Colletotrichum spp. on Strawberry Seedlings Enabling Forward Genetic Studies.

Generation and screening for nonpathogenic mutants is a popular tool for identifying pathogenicity-related genes. Successful application of this technique for plant fungal pathosystems requires reliable and rapid screening procedures. This study reports on the development of a rapid in vitro bioassay enabling large-scale screening and isolation of nonpathogenic mutants of Colletotrichum gloeosporioides and C. acutatum on strawberry seedlings. Inoculation was carried out on strawberry seedlings at two different developmental stages: 12-week-old (young) and 15-week-old (older) seedlings. A comparison was made between two inoculation techniques, (i) foliar dip and (ii) root soak, at two incubation temperatures (19 and 25°C). Mortality of young seedlings was observed 4 days after inoculation with both species, reaching 50% within 10 days, using both techniques at 25°C. However, mortality of older seedlings was delayed by 4 days compared with that in the young seedlings when using the root-soak method. Disease development decreased in young and older seedlings at the lower temperature. This method also was reliable in determining pathogenicity of the cucurbit-specific C. magna that did not cause disease symptoms on strawberry by either inoculation method. The proposed method enabled screening of more than 980 restriction enzyme-mediated integration mutants resulting in a selection of five reduced-virulence isolates. Initial characterization of some of these mutants revealed large differences in germination and appressorial formation compared with pathogenic isolates.

Differential protein expression in Colletotrichum acutatum: changes associated with reactive oxygen species and nitrogen starvation implicated in pathogenicity on strawberry.

The cellular outcome of changes in nitrogen availability in the context of development and early stages of pathogenicity was studied by quantitative analysis of two-dimensional gel electrophoresis of Colletotrichum acutatum infecting strawberry. Significant alterations occurred in the abundance of proteins synthesized during appressorium formation under nitrogen-limiting conditions compared with a complete nutrient supply. Proteins that were up- or down-regulated were involved in energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavenging. Members belonging to the reactive oxygen species (ROS) scavenger machinery, superoxide dismutase and glutathione peroxidase, were up-regulated at the appressorium formation stage, as well as under nitrogen-limiting conditions relative to growth with a complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressorium formation stage. Fungal ROS were detected within germinating conidia during host pre-penetration, penetration and colonization stages, accompanied by plant ROS, which were abundant in the apoplastic space. Application of exogenous antioxidants quenched ROS production and reduced the frequency of appressorium formation. Up-regulation in metabolic activity was detected during appressorium formation and nutrient deficiency compared with growth under complete nutrient supply. Enhanced levels of proteins related to the glyoxylate cycle and lipid metabolism (malate dehydrogenase, formate dehydrogenase and acetyl-CoA acetyltransferase) were observed at the appressorium formation stage, in contrast to down-regulation of isocitrate dehydrogenase. The present study demonstrates that appressoria formation processes, occurring under nutritional deprivation, are accompanied by metabolic shifts, and that ROS production is an early fungal response that may modulate initial stages of pathogen development.

A defect in nir1, a nirA-like transcription factor, confers morphological abnormalities and loss of pathogenicity in Colletotrichum acutatum.

SUMMARY A non-pathogenic mutant of Colletotrichum acutatum, designated Ca5, exhibited epiphytic hyphal growth and did not cause lesions on strawberry plants but grew necrotrophically when inoculated directly onto wounded stolons. In the absence of an external nitrogen source, the mutant exhibited extended germ-tube growth prior to appressorium formation. The deduced product of the impaired gene (nir1) is similar to NirA, an Aspergillus nidulans transcriptional regulator of nitrogen metabolism. Inoculation of leaves with wild-type or Ca5 conidia in the presence of a preferred nitrogen source resulted in massive epiphytic hyphal production, appressorium formation and rapid symptom development. Expression of C. acutatum wild-type nitrate reductase (nit1) and glutamine synthetase (gln1) was induced by nitrate but only nit1 expression was repressed in a rich medium. nit1 transcription increased during the appressorium-production stage, indicating that nitrogen starvation constitutes a cue for the regulation of appressorium development. The presence of nit1 transcript during various phases of infection is indicative of partial nitrogen starvation in planta. cAMP-dependent protein kinase A (PKA) was determined to be a negative regulator of immediate post-germination appressoria formation in the wild-type. As inhibition of PKA activity in the nir1 mutant did not affect appressoria formation, we suggest that NIR1 acts either in parallel or downstream of the PKA pathway. Our results show that nir1 is a pathogenicity determinant and a regulator of pre-infection development under nitrogen-starvation conditions and that nitrogen availability is a significant factor in the pre-penetration phase.