Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt.

In plants, long noncoding RNAs (lncRNAs) regulate disease resistance against fungi and other pathogens. However, the specific mechanism behind this regulation remains unclear. In this study, we identified disease resistance-related lncRNAs as well as their regulating genes and assessed their functions by infection of cotton (Gossypium) chromosome segment substitution lines with Verticillium dahliae. Our results demonstrated that lncRNA7 and its regulating gene Pectin methylesterase inhibitor 13 (GbPMEI13) positively regulated disease resistance via the silencing approach, while ectopic overexpression of GbPMEI13 in Arabidopsis (Arabidopsis thaliana) promoted growth and enhanced resistance to V. dahliae. In contrast, lncRNA2 and its regulating gene Polygalacturonase 12 (GbPG12) negatively regulated resistance to V. dahliae. We further found that fungal disease-related agents, including the pectin-derived oligogalacturonide (OG), could downregulate the expression of lncRNA2 and GbPG12, leading to pectin accumulation. Conversely, OG upregulated the expression of lncRNA7, which encodes a plant peptide phytosulfokine (PSK-α), which was confirmed by lncRNA7 overexpression and Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS) experiments. We showed that PSK-α promoted 3-Indoleacetic acid (IAA) accumulation and activated GbPMEI13 expression through Auxin Response Factor 5. Since it is an inhibitor of pectin methylesterase (PME), GbPMEI13 promotes pectin methylation and therefore increases the resistance to V. dahliae. Consistently, we also demonstrated that GbPMEI13 inhibits the mycelial growth and spore germination of V. dahliae in vitro. In this study, we demonstrated that lncRNA7, lncRNA2, and their regulating genes modulate cell wall defense against V. dahliae via auxin-mediated signaling, providing a strategy for cotton breeding.

Application of garlic allelochemicals improves growth and induces defense responses in eggplant (Solanum melongena) against Verticillium dahliae.

Aqueous garlic extracts (AGE) and garlic allelochemical diallyl disulfide (DADS) have been recently reported to bear bioactive properties to stimulate plant growth and development and alter defense-related physiology. We, therefore, performed a bioassay to study these chemicals as possible biostimulants for defense against Verticillium dahliae in eggplant seedlings. AGE and DADS were applied as a foliar application to the eggplants and samples were collected before and after pathogen inoculation at various intervals to analyze the defense mechanism. The obtained data revealed that with the application of AGE and DADS, the seedlings showed responses including activation of antioxidant enzymes, an abundance of chlorophyll contents, alteration of photosynthesis system, and accumulation of plant hormones compared to the control plants. Furthermore, the microscopic analysis of the AGE or DADS treated plants showed high variability in pathogen density within the root crown at 28 days post-inoculation. The low abundance of reactive oxygen species was noticed in AGE or DADS treated plants, which indicates that the plants were able to successfully encounter pathogen attacks. The AGE and DADS treated plants exhibited a lower disease severity index (32.4% and 24.8% vs 87.1% in controls), indicating successful defense against Verticillium infection. Our results were therefore among the first to address the biostimulatory effects of AGE or DADS to induce resistance in eggplant seedlings against V. dahliae and may be used to establish preparation for garlic-derived bioactive compounds to improve growth and defense responses of eggplants under-protected horticultural situations such as glasshouse or plastic tunnels system.

Verticillium isaacii is a Pathogen and Endophyte of Potato and Sunflower in the Columbia Basin of Washington.

The objective of this research was to test the hypothesis that Verticillium isaacii causes diseases of sunflower and potato plants. Two sunflower genotypes and one potato cultivar were inoculated with five V. isaacii isolates and three pathogenic V. dahliae isolates. Biomass, disease expression, and stem colonization were quantified. Overt wilt symptoms were observed on both sunflower genotypes and potato plants inoculated with a subset of the V. isaacii isolates. Biomass of both sunflower genotypes was not affected by V. isaacii infection. Tuber yields either decreased in response to infection by one V. isaacii isolate or were not affected by infection. Stems of sunflower and potato plants were infected by at least four of the five V. isaacii isolates. A new disease of sunflower and potato is documented. Evidence that V. isaacii exhibits different lifestyles including pathogenicity and endophytism is presented. Finally, this research documents variation in fungal lifestyles that can exist in samples from a single field.

Contrasting Patterns of Colonization with Verticillium longisporum in Winter- and Spring-Type Oilseed Rape (Brassica napus) in the Field and Greenhouse and the Role of Soil Temperature.

Oilseed rape, an important source of vegetable plant oil, is threatened by Verticillium longisporum, a soil-borne vascular fungal pathogen so far occurring in oilseed rape growing regions in Europe and Canada. Despite intensive research into V. longisporum in the last decades in controlled conditions, basic knowledge is still lacking about the time course of infection, temporal pattern of colonization, and disease development on field-grown plants. In this study, colonization of roots, stem bases, and stems with V. longisporum was followed by real-time PCR from the seedling until mature plant stages in 2-year field experiments with microsclerotia-infested plots and either spring-type or autumn-sown (winter-type) oilseed rape cultivars. The temporal pattern of plant colonization differed between greenhouse and field-grown oilseed rape and between spring- and winter-type plants in the field. Within 28 to 35 days, a continuous systemic colonization with V. longisporum was detected in roots and shoots of young plants in the greenhouse associated with significant stunting. In contrast, real-time PCR analysis of V. longisporum in field-grown winter oilseed rape plants displayed a strongly discontinuous colonization pattern with low fungal growth in roots during juvenile growth stages until flowering, whereas in spring oilseed rape, no root colonization was observed until early flowering stages. Hence, stem colonization with the pathogen required 6 months in winter oilseed rape and 1 month in spring oilseed rape from the time of initial root infection. The different patterns of stem colonization were related to soil temperature. Average soil temperatures in 5-cm depth during 7 days before sampling time points from 2 years of field experiments displayed a significant relationship with fungal colonization in the root. A climate chamber inoculation trial with soil temperature levels that varied from 6 to 18°C revealed a threshold temperature of >12°C in the soil to enable root invasion. This soil condition is reached in winter-type oilseed rape in the field in Germany either until the eight-leaf stage in early autumn or after pod stage in spring, whereas in spring-sown oilseed rape early root infection is delayed owing to the cool conditions during juvenile growth stages. The delay of stem colonization in field-grown oilseed rape may explain the lack of stunting as observed in the greenhouse and the previously reported inconsistent effects of V. longisporum on yield levels and seed quality, which were confirmed in this study.

Assessment of Resistance in Potato Cultivars to Verticillium Wilt Caused by Verticillium dahliae and Verticillium nonalfalfae.

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

Verticillium Wilt Caused by Verticillium dahliae and V. nonalfalfae in Potato in Northern China.

Potato (Solanum tuberosum L.) is one of the most important staple foods in many parts of the world including China. In recent years, Verticillium wilt has become a severe threat to potato production in China. During 2015 to 2016, 287 samples of symptomatic potato plants were collected from 15 counties in five provinces from northern China. One hundred and eighty-seven Verticillium-like colonies were isolated from these samples and identified to species based on cultural and morphological characteristics, and multigene phylogeny based on the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) genes. A consensus-rooted most parsimonious phylogenetic tree was generated from the data. One hundred and fifteen isolates comprising 61.5% of the total were identified as Verticillium dahliae, and the remaining 38.5% of the isolates were identified as V. nonalfalfae. V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mongolia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a single isolate). V. dahliae was not recovered from the samples in Ningxia. V. nonalfalfae dominated the collections from Gansu (87.2%) and Ningxia (100%) but was also recovered from Shaanxi (15.9%) and Inner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor V. alfalfae was recovered from the sampled areas. The V. nonalfalfae isolates were predominantly isolated from the samples collected from altitudes above 1,800 m, and in contrast, V. dahliae isolates were mainly recovered from fields sampled below 1,800 m. The optimum temperature for the colony growth of V. nonalfalfae was lower (20°C) than that for V. dahliae (25°C). Pathogenicity tests demonstrated that V. dahliae and V. nonalfalfae were both pathogens of potato Verticillium wilt, with V. dahliae isolates exhibiting higher virulence than V. nonalfalfae isolates regardless of the collection area of the species. This is the first documentation of V. nonalfalfae infecting S. tuberosum in China and the higher altitudes associated with infections of V. nonalfalfae anywhere in the world.

Molecular Dissection of Extracellular Matrix Proteome Reveals Discrete Mechanism Regulating Verticillium Dahliae Triggered Vascular Wilt Disease in Potato.

Plants exposed to patho-stress mostly succumb due to disease by disruption of cellular integrity and changes in the composition of the extracellular matrix (ECM). Vascular wilt, caused by the soil borne hemibiotrophic filamentous fungus Verticillium dahliae, is one of the most significant diseases that adversely affect plant growth and productivity. The virulence of the pathogen associated with the ECM-related susceptibility of the host plant is far from being understood. To better understand ECM-associated disease responses that allow the pathogen to suppress plant immunity, a temporal analysis of ECM proteome was carried out in vascular wilt susceptible potato cultivar upon V. dahliae infection. The proteome profiling led to the identification of 75 patho-stress responsive proteins (PSRPs), predominantly involved in wall hydration, architecture, and redox homeostasis. Two novel clues regarding wilt disease of potato were gained from this study. First, wall crosslinking and salicylic acid signaling significantly altered during patho-stress. Second, generation of reactive oxygen species and scavenging proteins increased in abundance leading to cell death and necrosis of the host. We provide evidence for the first time that how fungal invasion affects the integrity of ECM components and host reprogramming for susceptibility may function at the cell surface by protein plasticity.

Pathogen and Pest Responses Are Altered Due to RNAi-Mediated Knockdown of GLYCOALKALOID METABOLISM 4 in Solanum tuberosum.

Steroidal glycoalkaloids (SGAs) are major secondary metabolites constitutively produced in cultivated potato Solanum tuberosum, and α-solanine and α-chaconine are the most abundant SGAs. SGAs are toxic to humans at high levels but their role in plant protection against pests and pathogens is yet to be established. In this study, levels of SGAs in potato were reduced by RNA interference (RNAi)-mediated silencing of GLYCOALKALOID METABOLISM 4 (GAME4)-a gene encoding cytochrome P450, involved in an oxidation step in the conversion of cholesterol to SGA aglycones. Two GAME4 RNAi lines, T8 and T9, were used to investigate the effects of manipulation of the SGA biosynthetic pathway in potato. Growth and development of an insect pest, Colorado potato beetle (CPB), were affected in these lines. While no effect on CPB leaf consumption or weight gain was observed, early instar larval death and accelerated development of the insect was found while feeding on leaves of GAME4 RNAi lines. Modulation of SGA biosynthetic pathway in GAME4 RNAi plants was associated with a larger alteration to the metabolite profile, including increased levels of one or both the steroidal saponins or phytoecdysteroids, which could affect insect mortality as well as development time. Colonization by Verticillium dahliae on GAME4 RNAi plants was also tested. There were increased pathogen levels in the T8 GAME4 RNAi line but not in the T9. Metabolite differences between T8 and T9 were found and may have contributed to differences in V. dahliae infection. Drought responses created by osmotic stress were not affected by modulation of SGA biosynthetic pathway in potato.

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor.

Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.

Physiological response and sulfur metabolism of the V. dahliae-infected tomato plants in tomato/potato onion companion cropping.

Companion cropping with potato onions (Allium cepa var. agrogatum Don.) can enhance the disease resistance of tomato plants (Solanum lycopersicum) to Verticillium dahliae infection by increasing the expressions of genes related to disease resistance. However, it is not clear how tomato plants physiologically respond to V. dahliae infection and what roles sulfur plays in the disease-resistance. Pot experiments were performed to examine changes in the physiology and sulfur metabolism of tomato roots infected by V. dahliae under the companion cropping (tomato/potato onion). The results showed that the companion cropping increased the content of total phenol, lignin and glutathione and increased the activities of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase in the roots of tomato plants. RNA-seq analysis showed that the expressions of genes involved in sulfur uptake and assimilation, and the formation of sulfur-containing defense compounds (SDCs) were up-regulated in the V. dahlia-infected tomatoes in the companion cropping. In addition, the interactions among tomato, potato onion and V. dahliae induced the expression of the high- affinity sulfate transporter gene in the tomato roots. These results suggest that sulfur may play important roles in tomato disease resistance against V. dahliae.

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection.

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease-resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS-LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS-LRR disease-resistance proteins and triggers the production of trans-acting (ta)-siRNAs, most of which target mRNAs of defense-related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e-derived ta-siRNA-mediated silencing on NBS-LRR-disease-resistance proteins. It is speculated that a miR482-mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.

Alternative methods for the control of postharvest citrus diseases.

The postharvest diseases of citrus fruit cause considerable losses during storage and transportation. These diseases are managed principally by the application of synthetic fungicides. However, the increasing concern for health hazards and environmental pollution due to chemical use has required the development of alternative strategies for the control of postharvest citrus diseases. Management of postharvest diseases using microbial antagonists, natural plant-derived products and Generally Recognized As Safe compounds has been demonstrated to be most suitable to replace the synthetic fungicides, which are either being banned or recommended for limited use. However, application of these alternatives by themselves may not always provide a commercially acceptable level of control of postharvest citrus diseases comparable to that obtained with synthetic fungicides. To provide more effective disease control, a multifaceted approach based on the combination of different postharvest treatments has been adopted. Actually, despite the distinctive features of these alternative methods, several reasons hinder the commercial use of such treatments. Consequently, research should emphasize the development of appropriate tools to effectively implement these alternative methods to commercial citrus production.

Manipulating inoculum densities of Verticillium dahliae and Pratylenchus penetrans with green manure amendments and solarization influence potato yield.

We used cover crops with demonstrated efficacy against Verticillium dahliae and Pratylenchus penetrans in combination with the biocidal practice of solarization to determine the importance of targeting both organisms for managing potato early dying, an issue relevant to the search for alternatives to soil fumigation. Two experiments were conducted in commercial fields using a split-plot design with cover crop treatments of rapeseed, marigold, forage pearl millet, sorghum-sudangrass, and corn as the main plot factor and solarization as the subplot factor. Cover crops were grown and solarization applied in year one, followed by potato in year two. The main effect of solarization was significant for reduced inoculum levels of both organisms in year two and increased tuber yields. The main effect of cover crop was also significant with lower population densities of P. penetrans following the marigold and millet treatments and of V. dahliae following rape and sorghum-sudangrass. The cover crop treatments influenced yield in only one of the experiments in the absence of solarization. The combinatorial effect of cover crops and solarization resulted in a wide range of pathogen population densities. Mean soil inoculum levels were negatively related to yield for V. dahliae in experiment 1, and for P. penetrans and the P. penetrans × V. dahliae interaction in both experiments.

Purification and characterization of a CkTLP protein from Cynanchum komarovii seeds that confers antifungal activity.

BACKGROUND: Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic and antidiarrheal, but also as a herbal medicine to treat cholecystitis in people. We have found that the protein extractions from C. komarovii seeds have strong antifungal activity. There is strong interest to develop protein medication and antifungal pesticides from C. komarovii for pharmacological or other uses. METHODOLOGY/PRINCIPAL FINDINGS: An antifungal protein with sequence homology to thaumatin-like proteins (TLPs) was isolated from C. komarovii seeds and named CkTLP. The three-dimensional structure prediction of CkTLP indicated the protein has an acid cleft and a hydrophobic patch. The protein showed antifungal activity against fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea and Valsa mali. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed the transcription level of CkTLP had a significant increase under the stress of abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), NaCl and drought, which indicates that CkTLP may play an important role in response to abiotic stresses. Histochemical staining showed GUS activity in almost the whole plant, especially in cotyledons, trichomes and vascular tissues of primary root and inflorescences. The CkTLP protein was located in the extracellular space/cell wall by CkTLP::GFP fusion protein in transgenic Arabidopsis. Furthermore, over-expression of CkTLP significantly enhanced the resistance of Arabidopsis against V. dahliae. CONCLUSIONS/SIGNIFICANCE: The results suggest that the CkTLP is a good candidate protein or gene for contributing to the development of disease-resistant crops.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum.

Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC-MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.

Accelerated degradation of metam-sodium in soil and consequences for root-disease management.

We studied the development of accelerated degradation (AD) of methyl isothiocyanate (MITC) following repeated applications of its parent compound, metam-sodium (MS). Laboratory studies and four sets of field experiments were conducted during 2002-04 in three commercial fields in Israel. Repeated applications of MS to the three soils in the laboratory under controlled conditions demonstrated AD of MITC in some soils. In a peanut field, MS significantly reduced the incidence of Pythium pod rot and improved pod quality after a single application but its effectiveness was greatly reduced after two applications. In a second experiment, MS was significantly effective after a single application in controlling Verticillium wilt in potato but its efficacy diminished after three consecutive applications. In an additional experiment, fumigation with MS following single or double applications was more effective in reducing Verticillium wilt severity of potato compared with triple applications. Soils which did not develop AD of MITC were also recorded. Preplant MS fumigation of melon fields was effective at reducing sudden wilt following a single and two consecutive applications. Our study shows that development of AD of MITC might occur following repeated applications of MS in commercial fields. The data on MITC dissipation in soil following repeated MS applications under controlled conditions indicate the chemical's potential loss of activity under regular agricultural practices and the need for a management strategy to prevent such a development.

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants.

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.

Antimycotic activities of selected plant flora, growing wild in Lebanon, against phytopathogenic fungi.

Petroleum ether (PE) and methanolic extracts of nine wild plant species were tested in vitro for their antimycotic activity against eight phytopathogenic fungi. The efficacy of PE extracts against all pathogens tested was higher than that of methanolic extracts. Wild marjoram (Origanum syriacum) PE extract showed the highest and widest range of activity. It resulted in complete inhibition of mycelial growth of six of eight fungi tested and also gave nearly complete inhibition of spore germination of the six fungi included in the assay, namely, Botrytis cinerea, Alternaria solani, Penicillium sp., Cladosporium sp., Fusarium oxysporum f. sp. melonis, and Verticillium dahlia. The other plant extracts showed differential activities in the spore germination test, but none was highly active against mycelial growth. Inula viscosa and Mentha longifolia were highly effective (>88%) in spore germination tests against five of six fungi tested, whereas Centaurea pallescens, Cichorium intybus, Eryngium creticum, Salvia fruticosa, and Melia azedarach showed >95% inhibition of spore germination in at least two fungi. Foeniculum vulgare showed the least antimycotic activity. Fractionation followed by autobiography on TLC plates using Cladosporium sp. as a test organism showed that O. syriacum PE extracts contained three inhibition zones, and those of Inula viscosa and Cichorium intybus, two, whereas the PE extracts of the remaining plants showed each one inhibition zone. Some of the major compounds present in these inhibition zones were identified by GC-MS. The possibility for using these extracts, or their mixtures, to control plant diseases is discussed.