Selection of genetically diverse Trichoderma spp. isolates for suppression of Phytophthora capsici on bell pepper.

Environmentally compatible control measures are needed for suppression of Phytophthora capsici on pepper. Twenty-three isolates of Trichoderma were screened for suppression of a mixture of 4 genetically distinct isolates of this pathogen on bell pepper (Capsicum anuum) in greenhouse pot assays. Of these 23 isolates, GL12, GL13, and Th23 provided significant suppression of P. capsici in at least 2 assays. These isolates were then compared with Trichoderma virens isolates GL3 and GL21 for suppression of this disease in the presence and absence of the harpin-based natural product Messenger. Isolates GL3 and Th23 provided significant disease suppression (P ≤ 0.05) in 3 of 4 assays, while GL12, GL13, and GL21 provided significant suppression in 2 of 4 assays. There was no apparent benefit from the application of Messenger. Phylogenetic analysis of these 5 isolates (based on the ITS1 region of the nuclear rDNA cluster and tef1), and an additional 9 isolates that suppressed P. capsici in at least 1 assay, separated isolates into 2 clades, with 1 clade containing GL3, GL12, GL13, and GL21. There were also 2 more distantly related isolates, one of which was Th23. We report here the identification of genetically distinct Trichoderma isolates for potential use in disease management strategies employing isolate combinations directed at suppression of P. capsici on pepper.

Genetic and Biological Diversity of Trichoderma stromaticum, a Mycoparasite of the Cacao Witches'-Broom Pathogen.

ABSTRACT The witches'-broom disease, caused by the basidiomycete Crinipellis perniciosa, is the most limiting factor for cacao cultivation in Brazil. Trichoderma stromaticum is a mycoparasite of the witches'-broom pathogen of cacao that is currently being applied in the field to manage the disease in Bahia State, Brazil. In this work, molecular and traditional methods were used to study the genetic and biological diversity of this mycoparasite. Ninety-one isolates, mostly collected from farms not sprayed with the fungus, were analyzed by amplified fragment length polymorphisms (AFLP), which showed that two genetic groups (I and II) of T. stromaticum occur in Bahia State. This classification of T. stromaticum into two distinct AFLP groups was also in agreement with several other characteristics, including growth on agar media at different temperatures and sporulation on infected stem segments (broom pieces) and rice grains. Group II favors higher temperatures compared with group I. The genetic and biological differences of the isolates, however, were not evident in field experiments, where sporulation was evaluated on the surface of brooms under natural conditions. Our results show that there is considerable genetic and biological diversity within T. stromaticum in Bahia and other cacao-growing regions of South America that are affected by the witches'-broom disease. This diversity could be explored in the development of efficient biological control agents against the disease. Factors that may affect the application and performance of this biocontrol agent in the field, such as sporulation on rice substrate and on the brooms and growth at various temperatures, are discussed.

NEP1 orthologs encoding necrosis and ethylene inducing proteins exist as a multigene family in Phytophthora megakarya, causal agent of black pod disease on cacao.

Phvytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya.

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.

Effect of Formulated Plant Extracts and Oils on Population Density of Phytophthora nicotianae in Soil and Control of Phytophthora Blight in the Greenhouse.

Formulated plant extracts and oils were investigated for control of diseases caused by Phytophthora spp. Soil infested with chlamydospores of Phytophthora nicotianae was treated by incorporating 1, 5, and 10% aqueous emulsions of formulations containing clove oil, neem oil, pepper extract and mustard oil, cassia extract, synthetic cinnamon oil, or the fungicide metalaxyl. Population densities of P. nicotianae were determined at 0 (before treatment), 1, 3, 7, 14, and 21 days after treatment. Treatment of the soil with 5 and 10% aqueous emulsions resulted in significant (P < 0.05) differences among treatment mean values at each assay date. After 1 day, population densities were reduced to below the limit of detection (<0.04 CFU/cm3) in soil treated with 10% aqueous emulsions of two pepper extract-mustard oil formulations and two cassia extract formulations, and near the limit of detection for a synthetic cinnamon oil formulation. Over time, populations of P. nicotianae were detected in the assay; however, after 21 days, populations of P. nicotianae in soil treated with one of the pepper extract-mustard oil formulations still were not detected. Formulations of clove oil, another pepper extract-mustard oil combination, the two cassia extracts, and the synthetic cinnamon oil reduced populations 98.4 to 99.9% after 21 days compared with the nontreated control soil. The neem oil formulation and metalaxyl did not reduce pathogen populations at any rate tested. In the greenhouse after 35 days, 10% aqueous emulsions of a pepper extract-mustard oil formulation, a cassia extract, and the synthetic cinnamon oil formulation suppressed disease development in periwinkle 93.0 to 96.7% compared with the nontreated infested soil. The observed reduction in the pathogen population and significantly more healthy plants in the greenhouse indicates that these formulations of plant extracts and oils could have important roles in biologically based management strategies for control of diseases caused by P. nicotianae.

Effect of Botanical Extracts on the Population Density of Fusarium oxysporum in Soil and Control of Fusarium Wilt in the Greenhouse.

Several commercial formulations of botanical extracts and essential oils are being investigated as possible alternatives to soil fumigation for control of Fusarium wilt diseases. Soil infested with Fusarium oxysporum f. sp. chrysanthemi was treated with 1, 5, and 10% aqueous emulsions of formulated extracts of clove (70% clove oil), neem (90% neem oil), pepper/mustard (chili pepper extract and essential oil of mustard), cassia (extract of cassia tree), and Banrot (a standard fungicide applied at different labeled rates) in separate experiments. Population densities of F. oxysporum f. sp. chrysanthemi were determined at 0 (before treatment), 1, 3, 7, 14, and 21 days after treatment. Treatment of the soil with 5 and 10% aqueous emulsions resulted in significant (P < 0.05) differences among treatment means at each assay date. After 3 days, pepper/mustard, cassia, and clove extracts added as 10% aqueous emulsions reduced the population density of F. oxysporum f. sp. chrysanthemi 99.9, 96.1, and 97.5%, respectively, compared with the untreated control. Neem oil extract increased the population density of F. oxysporum f. sp. chrysanthemi at all concentrations tested. Banrot did not reduce the population density of F. oxysporum f. sp. chrysanthemi in any experiment. In a second, related experiment, soil infested with Fusarium oxysporum f. sp. melonis also was treated with 1, 5, and 10% aqueous emulsions of formulated extracts, incubated in closed plastic bags for 1 week, and planted with muskmelon seeds (cv. Gold Star) in the greenhouse. Treatment of infested soil with 5 and 10% aqueous emulsions of the botanical extracts resulted in differences among treatments after 5 to 6 weeks. The pepper/mustard, cassia, and clove extracts suppressed disease development in repeated experiments (80 to 100% healthy plant stand) compared with the untreated infested soil (<20% stand). The observed reduction in the pathogen population and increased healthy plant stand in the greenhouse indicates that these extracts could have important roles in biologically based management strategies for control of Fusarium wilt diseases.