Selection of Trichoderma stromaticum isolates for efficient biological control of witches’ broom disease in cacao

Witches’ broom is the most devastating disease of cacao in Brazil, and losses to it entail serious socio-economical and environmental problems. Biological control of the causal agent Moniliophthora perniciosa (Mp) using the antagonistic fungus Trichoderma stromaticum (Ts) is promising, although the identification of superior isolates is necessary. Here, we report a study on the selection of more effective Ts isolates based on field experiments. Sixty-three Ts isolates from a local collection were applied on brooms and placed under typical conditions of shaded-cacao plantations in southeastern Bahia State (Brazil), during two periods of three months each. The percentages of Ts sporulation and incidence and severity of Mp were the parameters used for biocontrol assessments. The results from both experiments were very distinct, indicating a high phenotypic variation in this collection and suggesting a significant effect of the environment in the Ts–Mp interaction. Ts-sporulation rates were negatively correlated with the presence of Mp in the brooms and a number of isolates reduced Mp incidence more efficiently than the reference isolate. Contrasting isolates in their efficiency of reducing Mp incidence were selected and further tested in four subsequent field trials for validation purposes. The results partially confirmed their biocontrol phenotypes but also suggested isolate-specific responses to environmental variations. Inhibition of Mp-basidiospore germination by total protein secreted in culture supernatants of Ts isolates correlated well with field results and revealed a potentially useful procedure for pre-screening of large collections towards selection of better biological control isolates. The characteristics and efficiency of the method as a reliable protocol for identification of superior BCAs in the witches’ broom—cacao pathosystem is discussed.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.     

Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America

Crinipellis perniciosa has been classified into at least four known biotypes associated with members of unrelated plant families. In this study, genetic variability is shown for 27 C (Cacao), 4 S (Solanum), and 7 L biotype (Liana) isolates of C. perniciosa collected from different regions of Brazil and South America. The objective was to investigate the genetic variability of the pathogen in the cacao-producing region of Bahia, Brazil, and elsewhere, through microsatellite analysis, and attempt to identify possible correlations between host specificity and electrophoretic karyotypes. The PCR-banding patterns were found to vary both within and between the different biotypes, and a correlation was established between the PCR-banding patterns and the chromosomal-banding patterns of each isolate. Microsatellite and chromosomal patterns among all of the L and S biotype isolates were distinctly different from the C biotypes analysed. A higher degree of genetic and chromosomal variability was found among C biotype isolates from the Amazon in comparison with C biotype isolates from Bahia, which seems to be comprised of only two main genotypes. This finding has important implications to the current cacao-breeding programme in Brazil.     

Trichoderma theobromicola and T. paucisporum: two new species isolated from cacao in South America

Trichoderma theobromicola and T. paucisporum spp. nov. are described. Trichoderma theobromicola was isolated as an endophyte from the trunk of a healthy cacao tree (Theobroma cacao, Malvaceae) in Amazonian Peru; it sporulates profusely on common mycological media. Trichoderma paucisporum is represented by two cultures that were obtained in Ecuador from cacao pods partially infected with frosty pod rot, Moniliophthora roreri; it sporulates sporadically and most cultures remain sterile on common media and autoclaved rice. It sporulates more reliably on synthetic low-nutrient agar (SNA) but produces few conidia. Trichoderma theobromicola was reintroduced into cacao seedlings through shoot inoculation and was recovered from stems but not from leaves, indicating that it is an endophytic species. Both produced a volatile/diffusable antibiotic that inhibited development of M. roreri in vitro and on-pod trials. Neither species demonstrated significant direct in vitro mycoparasitic activity against M. roreri.     

Development of novel microsatellites from Moniliophthora perniciosa, causal agent of the witches' broom disease of Theobroma cacao

Moniliophthora perniciosa is the causal agent of the witches' broom disease of cacao. Based on available genomic sequences, we identified 30 new microsatellite loci, which were analysed using 50 isolates from four populations sampled over a wide geographical area in Brazil, including three populations from the Amazon, the fungal putative centre of diversity, plus one from Bahia. Nine loci were polymorphic, with an average of 2.9 alleles per locus. The level of polymorphism observed was low, but these markers may allow the evaluation of pathogen diversity and the establishment of molecular standards for isolate fingerprinting to support cacao breeding.     

Production of lytic enzymes and siderophores, and inhibition of germination of basidiospores of Moniliophthora (ex Crinipellis) perniciosa by phylloplane actinomycetes

Streptomyces albovinaceus, Streptomyces caviscabies, Streptomyces griseus, Streptomyces setonii, and Streptomyces virginiae selected as antagonists of Moniliophthora (ex Crinipellis) perniciosa, the causal agent of cacao Witches’ broom, were examined in vitro to detect production of chitinases, β-1,3-glucanases, and cellulases. All the species produced chitinases, but not β-1,3-glucanases or cellulases, when grown on a liquid mineral medium containing glucose, colloidal chitin, or cell walls of M. perniciosa as a carbon source. There were no quantitative differences among species in the production of chitinase, however, the germination inhibition of basidiospores of M. perniciosa was higher when they were cultivated using glucose as a carbon source, followed by colloidal chitin and cell walls. All the species also produced hydroxymate type siderophores in similar quantities, and the quantity of siderophores did not correlate with the inhibition of basidiospore germination. The germination inhibition was more pronounced when S. albovinaceus, S. griseus, and S. virginiae were cultivated on iron-deficient medium, suggesting involvement of siderophores in the antagonism by these species of actinomycetes.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.     

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete Moniliophthora perniciosa, the causal agent of witches' broom in Theobroma cacao

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes witches' broom disease of Theobroma cacao. Analysis of the M. perniciosa draft genome led to the identification of three putative genes encoding necrosis and ethylene-inducing proteins (MpNEPs), which are apparently located on the same chromosome. MpNEP1 and 2 have highly similar sequences and are able to induce necrosis and ethylene emission in tobacco and cacao leaves. MpNEP1 is expressed in both biotrophic and saprotrophic mycelia, the protein behaves as an oligomer in solution and is very sensitive to temperature. MpNEP2 is expressed mainly in biotrophic mycelia, is present as a monomer in solution at low concentrations (<40 μm) and is able to recover necrosis activity after boiling. These differences indicate that similar NEPs can have distinct physical characteristics and suggest possible complementary roles during the disease development for both proteins. This is the first report of NEP1-like proteins in a basidiomycete.     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species

Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Distribution of assimilate during the flush cycle of growth in Theobroma cacao L.

The growth of the shoot and roots of seedling plants of cocoa (Theobroma cacao L.) under constant glasshouse conditions showed a rhythmic cycle, with the maximum growth stages of each alternating in a regular sequence. When the growth cycle of the shoot was upset by removing all new leaves immediately after unfolding, the roots showed a high constant growth rate during this period, suggesting that normally the rapidly expanding leaves exert an inhibitory influence on the roots. Conversely removal of portions of the root delayed the production of new leaves in the shoot. The level of soluble and starch carbohydrate in the mature leaves, roots and stem declined during the period of expansion of the flush leaves, but accumulated again at the end of the leaf expansion stage. It is likely that this reserve carbohydrate was remobilised and translocated to the flush leaves during their period of expansion. A large proportion of newly formed photoassimilate, as shown by the distribution of ¹⁴C radioactivity from different source leaves, was also translocated to the young leaves during expansion. The large sink created by these leaves may cause photoassimilate and reserve carbohydrate to be diverted from the roots, thereby inhibiting root growth during the stage of leaf expansion. It is suggested that the rhythmic leaf production at the apex may control the growth cycle of the roots.     

Isolation of ESTs from cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) leaves treated with inducers of the defense response

Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma cacao L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecules such as salicylic acid, jasmonic acid and ethylene. Activation of plant defenses is associated with changes in the expression of large numbers of genes. To gain a better understanding of defense responses in cacao, we have employed suppressive subtractive hybridization (SSH) cDNA libraries, macroarray hybridization analysis, high throughput DNA sequencing and bioinformatics to identify cacao genes induced by these signaling molecules. Additionally, we investigated gene activation by a phytotoxic elicitor-like protein, Nep1. We have identified a unigene set of 1,256 members, including 330 members representing genes induced during the defense response.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Sequences presented here are deposited with GenBank under accession numbers CF972636–CF974749