dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

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.     

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.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

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.     

Correlation between screening procedures to select root endophytes for biological control of Fusarium verticillioides in Zea mays L

Biological control is an alternative to pesticides for protection against crop diseases. A key to progress in the field of biological control to protect maize against Fusarium verticillioides is to select in vitro the best agent to be applied in the field. This research was undertaken to evaluate the correlation between different screening methods and to suggest an adequate procedure that could be used to select bacterial agents with potential biocontrol against F. verticillioides in the maize rhizosphere. The results show an extensive Pearson correlation coefficient analysis between different screening procedures carried out for Arthrobacter spp., Azotobacter spp., Pseudomonas spp., and Bacillus spp. paired with 13 F. verticillioides strains isolated from maize endorhizosphere. The following screening methodologies were correlated: niche overlap index (NOI), indices of dominance, growth rate, lag phase, antibiosis, and fumonisin production. It was observed that NOI and antibiosis methodologies did not show any correlation. They were used to choose the best bacteria to apply under greenhouse conditions. Azotobacter armeniacus RC2 showed a NOI > 0.9 and inhibited all F. verticillioides strains assayed. Seed maize bacterization with A. armeniacus RC2 at 10⁶ and 10⁷ inoculum level resulted in significantly lower values of F. verticillioides counts compared to the control without bacteria at the root levels assayed. The analysis of variance indicated that there were significant interactions between two fungal repression assays. Antibiosis assays significantly correlated with greenhouse conditions (p < 0.01) at two inoculum levels tested. Therefore, the selection system adopted was adequate to choose the best bacterial biocontrol agent. The application of this methodology shows a way to outline the best biocontrol candidate.     

Screening rhizobacteria for biological control of Ralstonia solanacearum in Ethiopia

Bacterial wilt caused by Ralstonia solanacearum (Smith) has become a severe problem mainly on potato and tomato in Ethiopia and no effective control measure is available yet. To explore possibilities for the development of biological control for the disease, 118 rhizobacteria, most of them collected from Ethiopia, were screened against an Ethiopian R. solanacearum strain. On the basis of in vitro screening, six strains (RP87, B2G, APF1, APF2, APF3, and APF4) with good inhibitory effect were selected for in planta testing in a greenhouse. In the greenhouse, soil and tomato seedlings were treated with the antagonists and their effects studied. The study showed that APF1 and B2G strains significantly reduced disease incidence and increased weight of tomato plants. Area under disease progress curves (AUDPC) was reduced by 60% and 56% in plants inoculated with APF1 and B2G strains, respectively. Plant dry weight increase in plants inoculated with APF1 and B2G strains was 96% and 75%, respectively. APF1 was found to be the most beneficial strain in disease suppression and also growth promotion resulting in 63% dry weight increase compared to untreated control. The study revealed that APF1 and B2G strains are promising strains whose effectiveness under field conditions and their mode of action should be investigated.     

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.     

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 .     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

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.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

Effect of pollination intensity on fruit and seed set in cacao (Theobroma cacao L.)

We studied the functional relationship between pollination intensity and fruit survival as well as the number of seeds per pod in the tropical tree Theobroma cacao L. on a Forastero Upper-Amazon clone (UPA 409) in Ivory Coast. Cutting the style 24 h after pollination allowed for counting the number of pollen grains deposited on a stigma without affecting fruit set and seed development. Forty-three pollen grains were necessary to reach 50% of maximum fruit set 28 days after pollination. Above 115 pollen grains, the proportion of developing ovaries reached a maximum of 88% 28 days after pollination and 75% at maturity. With fewer than 238 pollen grains per stigma, there was a close relationship between pollination intensity and number of seeds per pod; the pollenseed ratio increased from 1.61 to 3.81 for PI increasing from 30 to 238 pollen grains. For higher pollination intensities, the average number of seeds per pod reached a maximum of 58. The relationship between pollination intensity and seed content was modelled. Results are consistent with the hypothesis that ovules attracted pollen tubes in a similar way regardless of whether or not they had already been reached by another pollen tube.     

Mapping of Quantitative Trait Loci for Butter Content and Hardness in Cocoa Beans (Theobroma cacao L.)

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F₂ population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.     

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     

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.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

Effects of VA mycorrhiza on the growth of cacao seedlings under nursery conditions in Venezuela

The effects of vesicular-arbuscular mycorrhizae inoculation on the growth of cacao seedlings (Theobroma cacao var. Ocumare 60) grown for 5 months in a nursery were studied. The effects of introduced VAM fungi (Glomus occultum, Acaulospora appendicula, Glomus manihotis, Acaulospora morrowae and Scutellospora pellucida) in soils treated with copper oxychloride or methyl bromide, were compared with the indigenous VAM and with their respective non-inoculated controls.Cacao seedlings responded well to indigenous VAM fungi, which included Scutellospora calospora as the dominant species, inducing increases significant in height, dry weight and foliar uptake of P, Cu and Zn in relation to the sterile control. G. occultum and A. appendicula increased the height of cacao seedlings but to an extent not yet statistically significant through the duration of the experiment. S. pellucida and A. appendicula doubled the phosphorus uptake of cacao seedlings. The methyl bromide sterilization induced the lowest Cu and Zn uptake in the cacao seedlings but this effect was ameliorated with VAM inoculation. Manifest deficiency symptoms were not observed in the treatments.Copper oxychloride treatment depressed growth to the same level as the sterile control although its residual effects did not kill VAM. It could change the competitive relations among the VAM species and in this case seemed to affect adversely the more efficient native fungi.The influence of the presence and long permanence of large cotyledons in cacao on the results obtained is discussed.