Genetic Differentiation of Verticillium dahliae Populations Recovered from Symptomatic and Asymptomatic Hosts.

Verticillium dahliae is a soilborne fungal pathogen affecting many economically important crops that can also infect weeds and rotational crops with no apparent disease symptoms. The main research goal was to test the hypothesis that V. dahliae populations recovered from asymptomatic rotational crops and weed species are evolutionarily and genetically distinct from symptomatic hosts. We collected V. dahliae isolates from symptomatic and asymptomatic hosts growing in fields with histories of Verticillium wilt of potato in Israel and Pennsylvania (United States), and used genotyping-by-sequencing to analyze the evolutionary history and genetic differentiation between populations of different hosts. A phylogeny inferred from 26,934 single-nucleotide polymorphisms (SNPs) in 126 V. dahliae isolates displayed a highly clonal structure correlated with vegetative compatibility groups, and isolates grouped in lineages 2A, 2B824, 4A, and 4B, with 77% of the isolates in lineage 4B. The lineages identified in this study were differentiated by host of origin; we found 2A, 2B824, and 4A only in symptomatic hosts but isolates from asymptomatic hosts (weeds, oat, and sorghum) grouped exclusively within lineage 4B, and were genetically indistinguishable from 4B isolates sampled from symptomatic hosts (potato, eggplant, and avocado). Using coalescent analysis of 158 SNPs of lineage 4B, we inferred a genealogy with clades that correlated with geographic origin. In contrast, isolates from asymptomatic and symptomatic hosts shared some of the same haplotypes and were not differentiated. We conclude that asymptomatic weeds and rotational hosts may be potential reservoirs for V. dahliae populations of lineage 4B, which are pathogenic to many cultivated hosts.

Postharvest dark skin spots in potato tubers are an oversuberization response to Rhizoctonia solani infection.

Israeli farmers export 250,000 tons of potato tubers annually, ≈40,000 tons of which are harvested early, before skin set. In recent years, there has been an increase in the occurrence of dark skin spots on early-harvested potato tubers ('Nicola') packed in large bags containing peat to retain moisture. The irregular necrotic spots form during storage and overseas transport. Characterization of the conditions required for symptom development indicated that bag temperature after packing is 11 to 13°C and it reaches the target temperature (8°C) only 25 days postharvest. This slow decrease in temperature may promote the establishment of pathogen infection. Isolates from typical lesions were identified as Rhizoctonia spp., and Koch's postulates were completed with 25 isolates by artificial inoculation performed at 13 to 14°C. Phylogenetic analysis, using the internal transcribed spacer sequences (ITS1 and ITS2) of rDNA genes, assigned three isolates to anastomosis group 3 of Rhizoctonia solani. Inoculation of wounded tubers with mycelium of these R. solani isolates resulted in an oversuberization response in the infected area. With isolate Rh17 of R. solani, expression of the suberin biosynthesis-related genes StKCS6 and CYP86A33 increased 6.8- and 3.4-fold, respectively, 24 h postinoculation, followed by a 2.9-fold increase in POP_A, a gene associated with wound-induced suberization, expression 48 h postinoculation, compared with the noninoculated tubers. We suggest that postharvest dark spot disease is an oversuberization response to R. solani of AG-3 infection that occurs prior to tuber skin set.

Management of Verticillium Wilt of Avocado Using Tolerant Rootstocks.

The global avocado industry is growing, and farmers are seeking to expand their plantations. However, many lands suitable for avocado planting were previously cultivated with hosts of the soil-borne fungal pathogen Verticillium dahliae, which is the causal agent of Verticillium wilt (VW). VW can seriously impair avocado orchards, and therefore, planting on infested soil is not recommended. The use of different rootstock types allows avocado cultivation in various regions with diverse biotic and abiotic constraints. Hence, we tested whether genetic variance among rootstocks may also be used to manage avocado VW. Six hundred trees, mostly Hass and some Ettinger, grafted on 23 selected rootstocks were evaluated for five years in a highly V. dahliae-inoculated plot for VW symptoms, fungal infection, and productivity. The selected rootstocks displayed a significant variation related to VW tolerance, and productive avocado rootstocks with potential VW tolerance were identified. Moreover, the rootstock productivity appears to correlate negatively to the susceptibility level. In conclusion, planting susceptible rootstocks (e.g., VC66, VC152, and VC26) in infested soil increases the likelihood of massive tree loss and low productivity. Whereas, tolerant rootstocks (e.g., VC804 and Dusa) may restrict VW and enable avocado cultivation on infested soils.

Discrimination of soil-borne fungi using fourier transform infrared attenuated total reflection spectroscopy.

Fourier transform infrared (FT-IR) attenuated total reflection (ATR) spectroscopy was used to discriminate five commonly encountered soil-borne fungi that cause severe economic damage to agriculture: Colletotrichum, Fusarium, Pythium, Rhizoctonia, and Verticillium. Contrary to previous studies related to microorganism discrimination using FT-IR-ATR spectroscopy, the pathogen samples were not dried on the ATR crystal, which is a time-consuming operation. Rather, after removing some pathogen filaments from the solution using tweezers, these were placed directly on a flat ATR crystal and pressure was applied using a pressure clamp. Following water subtraction, baseline correction, and normalization of the spectra, principal component analysis was used as a data-reduction step and canonical variate analysis was used for discrimination. Discrimination was performed at the genus level and at the strain level for Colletotrichum. For discrimination between the five fungi at the genus level, the success rate for the validation samples ranged from 75% to 89%. For discrimination between the two Colletotrichum strains, the success rate was 78%. Comparison with spectra of similar fungi dried on the ATR crystal showed that both types of spectra were very similar, indicating that drying the samples on the ATR crystal is not required and can be replaced by mathematical post-processing of the spectra. For routine analyses that involve rapid screening of very large amounts of samples, this approach allows for increasing significantly the number of samples that can be analyzed daily.

Early and rapid detection of potato's fungal infection by Fourier transform infrared microscopy.

Fungi are considered serious pathogens to many plants and can cause severe economic damage. Early detection of these pathogens is very important and might be critical for their control. The available methods for detection of fungi are time consuming and not always very specific. Fourier transform infrared (FT-IR) microscopy has proved to be a reliable and sensitive method for detection of molecular changes in cells. Fungi pathogens display typical infrared spectra that differ from the spectra of substrate material such as potato. In the present study we used FT-IR microscopy for early and rapid detection of the potato fungal pathogen Colletotrichum coccodes on the surface of potato tubers. Infected potatoes with this fungal pathogen and uninfected potatoes were examined and correctly classified as infected or not infected by FT-IR microscopy at very early stages of infection when no morphological signs of infection could be seen. Unique spectral biomarkers were found in naturally infected potatoes compared to disease-free control potatoes.

Effect of Colletotrichum coccodes on Potato Yield, Tuber Quality, and Stem Colonization During Spring and Autumn.

The response of five potato cultivars to Colletotrichum coccodes was tested in artificially inoculated fields for three consecutive spring and autumn seasons during 1994 to 1996. Significant yield reductions (22 to 30%) were observed in all tested cultivars. Results varied between years, but yield losses were more severe in autumn than in spring. Stem infections of plants were observed 90 days after planting on the surface of the stem and in vascular tissue. C. coccodes inoculation also resulted in reduction of the quality of daughter tubers. Cultivars Cara and Nicola were found to be less susceptible to tuber infection than Alpha, Desiree, and Agria. The incidence of diseased daughter tubers was higher when the soil was infested than when the foliage was inoculated. C. coccodes contamination of dry stems at harvest (in inoculated plots) was relatively high in all cultivars, with no difference between inoculation methods. Thus, C. coccodes infection not only affects potato yield and the quality of potatoes for seed and consumption, but also contaminates soil and serves as an important source of inoculum for future potato crops.

Verticillium Wilt of Paprika Caused by a Highly Virulent Isolate of Verticillium dahliae.

Verticillium dahliae caused wilting, stunting and early dying of paprika plants in Israel, resulting in a 22% reduction in yield. A V. dahliae isolate from diseased paprika plants was slower growing on agar medium than V. dahliae isolates from potato, tomato, watermelon, and Dodonaea, but formed microsclerotia earlier. The paprika isolate of V. dahliae was highly virulent to paprika in pathogenicity tests. Disease severity indices obtained from three paprika cultivars ranged from 3.7 to 4.9 (on a scale of 0 to 5). The disease caused height reductions of 43 to 62% in the three inoculated paprika cultivars, and pathogen colonization levels were 10- to 35-fold higher in stems than in leaves. The potato isolate of V. dahliae did not produce symptoms or cause stunting of paprika, and could not be detected in stems or leaves. The tomato, watermelon, and Dodonaea isolates did not cause disease symptoms in paprika, although they colonized the plant tissue. In potato and eggplant, the paprika isolate caused a higher incidence of disease with more severe symptoms and a higher level of tissue colonization than did the potato isolate.

Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60.

Dickeya species relatedness and clade structure determined by comparison of recA sequences.

Using sequences from the recA locus, we have produced a phylogeny of 188 Dickeya strains from culture collections and identified species relatedness and subspecies clade structure within the genus. Of the six recognized species, Dickeya paradisiaca, D. chrysanthemi and D. zeae were discriminated with long branch lengths. The clade containing the D. paradisiaca type strain included just one additional strain, isolated from banana in Colombia. Strains isolated from Chrysanthemum and Parthenium species made up most of the clade containing the D. chrysanthemi type strain, and the host range of this species was extended to include potato. The D. zeae clade had the largest number of sequevars and branched into two major sister clades that contained all of the Zea mays isolates, and were identified as phylotypes PI and PII. The host range was increased from six to 13 species, including potato. The recA sequence of an Australian sugar-cane strain was sufficiently distinct to rank as a new species-level branch. In contrast to these species, Dickeya dadantii, D. dianthicola and D. dieffenbachiae were distinguished with shorter branch lengths, indicating relatively closer relatedness. The recA sequence for the type strain of D. dadantii clustered separately from other strains of the species. However, sequence comparison of three additional loci revealed that the D. dadantii type strain grouped together with the six other D. dadantii strains that were sequenced. Analysis of all four loci indicated that the D. dadantii strains were most closely related to D. dieffenbachiae. Three further branches (DUC-1, -2 and -3) were associated with these three species, which all diverged from a common origin and can be considered as a species complex. The large clade containing the D. dianthicola type strain comprised 58 strains and had little sequence diversity. One sequevar accounted for the majority of these strains, which were isolated nearly exclusively from eight hosts from Europe. Isolation of this sequevar on multiple occasions from Dianthus and (more recently) potato demonstrates that this lineage has become established in these species. The D. dadantii clade comprised 11 sequevars, and the known host range of the species was extended from eight to 19 species. New hosts included several ornamental species and potato. The clade DUC-1 was made up exclusively of potato strains originating from Europe, which had identical sequences, whilst DUC-2 strains were isolated mostly from a variety of monocotyledonous species. A single strain from Aglaonema sp. made up DUC-3. A single sequevar constituted the D. dieffenbachiae clade. The phylogenetic method described will provide a simple means for identification to the species and intraspecies level, which will support efforts to control these pathogens based on monitoring and surveillance.