Comparative analysis of temporal transcriptome reveals the relationship between pectin degradation and pathogenicity of defoliating Verticillium dahliae to Upland cotton (Gossypium hirsutum)

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is a major plant disease that causes heavy annual losses around the world, especially in Upland cotton (Gossypium hirsutum). The disease-causing pathogen can be classified into defoliating (D) and non-defoliating (ND) pathotypes based on the induced symptoms. At present, little is known about the complex mechanisms of fungal pathogenicity and cotton resistance to it. Comparative analysis of temporal transcriptome was performed on two V. dahliae strains, Vd_086 (D) and Vd_BP2 (ND), at key development stages (hyphal growth, microsclerotia production, and spore germination) to reveal the functional process on plant defoliation and death. Differentially expressed gene (DEG) analysis revealed a strong correlation between cell wall protein kinase activities and the early pathogenicity of defoliating Vd_086. With weighted gene co-expression network analysis (WGCNA), six specific gene modules were correlated with the biological traits of the fungal samples. Functional enrichment with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways together with DEG analysis revealed six pectin degrading enzymes including Polygalacturonase gene 1 (PG1), Pectate lyase gene (PEL) and Pectinesterase gene 1 (PME1) expressed in the early development of Vd_086 that may be related to the robust pathogenicity of this strain during the early invasion. The expression of four of these genes was verified by real-time quantitative reverse transcription PCR (qRT-PCR). In addition, we identified Mitogen-Activated Protein Kinase (MAPK) signaling “hub” genes that may regulate these pectinases. In a word, enhanced expression of pectin degradation enzymes is associated with the stronger pathogenicity of Vd_086 than Vd_BP2, especially at early infection stages. The disease-causing capability is likely regulated by MAPK signaling genes. This study provides new insight into molecular mechanisms of the plant-pathogen interaction on the VW disease, facilitating more effective control measures against this pathogen, including molecular breeding for the VW-resistant cotton cultivars.Author summaryVerticillium wilt (VW), caused by fungal pathogen Verticillium dahliae (Vd), is arguably the most devastating disease in cotton production for decades. Molecular biologists and plant breeders have been working hard to identify host plant resistant genes for many years but have met with little success due to the large complex genome of cotton. The V. dahliae strains are grouped in two pathotypes, of which defoliating (D) strains cause total leaf loss of infected cotton plants and non-defoliating (ND) strains do not. Comparative transcriptome analysis of D strain Vd_086 and ND strain Vd_BP2 identified the candidate genes and molecular mechanisms related to the Vd pathogenicity. Besides the difference in pathogenicity, these strains are distinguishable by the rate of hyphal elongation, microsclerotia production, and spore germination. With these phenotypes, transcriptome sequencing of both strains was performed at the three growth phases. By the combination of comparative transcriptomic differentially expressed gene (DEG) analysis and weighted gene correlation network analysis (WGCNA), cell wall-associated pectinase genes were found to be active at hyphal elongation stage of the V. dahliae pathogen and ribosome-related processes were activated for microsclerotia production. Gene modification processes were activated with many protein kinases at spore germination stage that for the next infection cycle. Furthermore, four pectinases in the pentose and glucuronate interconversion (PGI) pathway were identified and verified as highly expressed in the D strain with strong pathogenicity to Upland cotton (Gossypium hirsutum). Our results provided evidence in support of the hypothesis that stronger early pathogenicity of the D strain is resulted from greater plant cell wall pectin degradability. Transcription factors (TFs) and “hub” module genes were identified in searching of protein interaction for possible regulators of the recognized pectinases. TFs involved in mitogen-activated protein kinase (MAPK) signaling pathway were shown to regulate not only hyphal processes but also the entire growth period of V. dahliae. This is the first study known to use module extraction techniques of WGCNA to identify differentially co-expressed genes between two fungal pathotypes of V. dahliae strains. The study provides new insights into molecular mechanisms of the plant-pathogen interaction and may lead to molecular breeding for resistant cotton cultivars to effectively control this devastating disease.

Liquid culture production of microsclerotia of Purpureocillium lilacinum for use as bionematicide

The production of microsclerotia by Purpureocillium lilacinum in liquid culture for use as a biocontrol agent for management of root-knot nematode, Meloidogyne incognita, has not been described. To investigate the potential for microsclerotia production, P. lilacinum strain CQPL01 was cultured in liquid media containing various concentrations of ferrous sulphate. Under these conditions mycelia began to form microsclerotia. The maximum yield (11.8 × 104 microsclerotia ml−1) was obtained in medium containing 0.2 g l−1 ferrous sulphate and the greatest production of conidia (1.3 × 108 conidia g−1) was obtained by the culture of air-dried microsclerotia in aqueous agar medium. Subsequently, the viability of microsclerotia, including stress resistance, storage stability and pathogenicity against M. incognita, was investigated. The microsclerotia exhibited excellent nematophagous ability and greater thermotolerance and UV-B radiation tolerance compared to conidia. These results suggested that microsclerotia propagules might be superior to the use of conidia in P. lilacinum biocontrol products.

Disease Severity and Microsclerotium Properties of the Sorghum Sooty Stripe Pathogen, Ramulispora sorghi

Ramulispora sorghi causes sooty stripe of sorghum. Disease severity in irrigated and dryland plots was measured for 25 susceptible sorghum genotypes during the 2007 and 2008 growing seasons using a rating scale based upon percent leaf area infected. Disease severity ratings were approximately 1.4 points higher (P < 0.0001) on the rating scale in the irrigated plots than dryland plots for 2007 and 2008. Sooty stripe lesions were collected from each sorghum genotype in irrigated plots and assessed for mean microsclerotium production within lesions, microsclerotium size, and sporogenic germination, with significant differences apparent between genotypes for microsclerotium size (P = 0.01) and sporogenic germination (P = 0.01). There was no relationship between disease severity and microsclerotium production within leaf lesions, microsclerotium size, or sporogenic germination; however, there was a positive and significant correlation between microsclerotia production within a lesion and microsclerotium size (R2 = 0.19, P < 0.0001). Although microsclerotia from sorghum lesions varied in structural characteristics and their ability to produce spore masses, these qualities were dependent upon the sorghum genotype from which the microsclerotia were derived, because the R. sorghi population was genetically uniform as determined by internal transcribed spacer sequences and random amplified polymorphic DNA polymerase chain reaction.

A Study of Environmental Factors that Affect Survival of Pumpkin Isolates of Verticillium dahliae

V. dahliae is a destructive soilborne pathogen to many economically important crops worldwide. Knowledge of environmental influences, including temperature, source, and availability of nutrients and pH, on disease development is central to devising control strategies. The effects of root decoction media, pH, and temperature on radial growth and microsclerotia production in 32 Verticillium dahliae (Kleb.) isolates from Trinidad were investigated. There were significant differences (P ≤ 0.05) in colony diameters and growth rates for isolates grown on different media. The highest growth occurred on 5% nutsedge root extract agar (NREA) and the least amount of growth was obtained on 1% pumpkin root extract agar (PREA). Production of microsclerotia was significantly higher on 5% PREA, 5% NREA, and potato dextrose agar (PDA). Growth tests on PDA formulated to different pH levels revealed that the most growth occurred at pH 5.2, but this was not significantly different from colony diameters obtained at pH 4.2, 6.2, and 7.2. pH, however, had a pronounced effect on production of microsclerotia. The optimum temperature for radial growth and formation of microsclerotia in V. dahliae in vitro was found to be 25 ± 1 °C. The effects of isolates and temperature on incidence and symptom severity in susceptible pumpkin plants were also tested under controlled conditions. There was no significant difference in symptom severity when inoculated plants were grown at 25 ± 1 °C and at 30 ± 1 °C. The pathogen was recovered from infected plants grown at 35 ± 1 °C, although growth is completely inhibited at this temperature in vitro. There was no significant difference among isolates in their pathogenic response at the four different temperatures tested.

Herbicide Effects onRhizoctonia solaniin Vitro and Rhizoctonia Foliar Blight of Soybean (Glycine max)

Acifluorfen, alachlor, glufosinate, glyphosate, paraquat, and pendimethalin were evaluated for their effects on mycelial growth and sclerotia/microsclerotia production byRhizoctonia solaniAG-1 IA and IB in culture. All of these herbicides except glufosinate and glyphosate were evaluated for effects on severity of Rhizoctonia foliar blight of soybean in the field. In laboratory studies, all herbicides reduced colony radius ofR. solani.Growth reductions for IB were greater than for IA in the presence of pendimethalin, alachlor, and acifluorfen, but glufosinate reduced growth of IA more than IB. Sclerotia production by both isolates was prevented by paraquat, greatly reduced by glufosinate, but markedly less affected by the other herbicides tested. In field studies, all tested herbicides influenced severity of Rhizoctonia foliar blight when disease pressure was low, but only paraquat reduced severity when disease pressure was high.

Pyrenochaeta terrestris Microsclerotia Production and Pigmentation on Onion Roots

Experiments were conducted to determine variation of in vitro microsclerotia production, pigmentation, and growth of five Pyrenochaeta terrestris (Hans.) Gorenz, Walker, and Larson isolates from Texas and New Mexico. Isolates of P. terrestris, the causal agent of pink root rot, were placed on agar with lo-mm-long sections of sterile onion (Allium cepa L.) roots. Microsclerotia were present after 20 days at 20, 25, 27, or 32C, with the number of microsclerotia dependent on the isolate. Microsclerotia were absent at 35C. One isolate produced few microsclerotia at all temperatures. Optimum temperatures for growth on potato dextrose agar for the isolates tested were 25 and 27C. Pigment production in roots on agar varied depending on isolate and temperature. Three isolates produced high levels of pigment in onion roots at 15, 20, 25, and 27C. A New Mexico isolate produced significantly less visible pigment than the other New Mexico and Texas isolates. One isolate produced very little pigment at all temperatures tested. Constant fluorescent light stimulated pycnidia production in one isolate and reduced microsclerotia production in all other isolates. Isolates varied significantly in microsclerotia production and pigment synthesis.

MORPHOGENESIS IN VERTICILLIUM: EFFECTS OF LIGHT AND ULTRAVIOLET RADIATION ON MICROSCLEROTIA AND MELANIN

Delay or total suppression of microsclerotia and melanin formation by light seems general among Verticillium isolates of the microsclerotial form. White, fluorescent light delayed or prevented production of microsclerotia and melanin in 14 of 16 microsclerotial isolates on agar, and in 30 of 31 isolates which produced at least some microsclerotia and melanin in liquid culture. Most isolates produced more melanin and microsclerotia on a complex medium (potato dextrose agar) than in a defined medium (sucrose–nitrate liquid). Near-ultraviolet radiation (peak 3650 Å) suppressed melanin and microsclerotia production in one isolate (H-13) of Verticillium in both liquid and solid media. On agar, colonies of isolate H-13 grew larger in near-UV than in darkness. Isolate H-13 retained its ability to produce melanin and microsclerotia during 21 days suppression by near-UV. On some media, near-UV inhibits all steps in microsclerotia development. On others it inhibits only the final steps. There appear to be at least two places in the chain of events leading to melanin and microsclerotia production where near-UV can inhibit the process.