Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches' Broom Disease

The basidiomycete fungus Crinipellis perniciosa (Stahel) Singer is the causal agent of Witches' Broom Disease of Cacao (Theobromacacao L.) which is the main factor limiting cacao production in the Americas. Pod losses of up to 90% are experienced in affected areas as evidenced by the 50% drop in production in Bahia province, Brazil following the arrival of the C. perniciosa in the area in 1989. The disease has proven particularly difficult to control and many farmers in affected areas have given up cacao cultivation. In order to evaluate the potential of endophytes as a biological control agent of this phytopathogen, the endophytic fungal community of resistant and susceptible cacao plants as well as affected branches was studied between 2001 and 2002. The fungal community was identified by morphological traits and rDNA sequencing as belonging to the genera Acremonium, Blastomyces, Botryosphaeria, Cladosporium, Colletotrichum, Cordyceps, Diaporthe, Fusarium, Geotrichum, Gibberella, Gliocladium, Lasiodiplodia, Monilochoetes, Nectria, Pestalotiopsis, Phomopsis, Pleurotus, Pseudofusarium, Rhizopycnis, Syncephalastrum, Trichoderma, Verticillium and Xylaria. These fungi were evaluated both in vitro and in vivo by their ability to inhibit C. perniciosa. Among these, some were identified as potential antagonists, but only one fungus (Gliocladium catenulatum) reduced the incidence of Witches' Broom Disease in cacao seedlings to 70%.     

Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa

BACKGROUND AND AIMS: Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. METHODS: Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. KEY RESULTS: A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. CONCLUSIONS: As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.     

Infection Biology of Moniliophthora perniciosa on Theobroma cacao and Alternate Solanaceous Hosts

The C-biotype of Moniliophthora perniciosa is the causal agent of witches’ broom disease of Theobroma cacao L. While this disease is of major economic importance, the pathogenicity mechanisms and plant responses underlying the disease are difficult to study given the cacao tree’s long life cycle and the limited availability of genetic and genomic resources for this system. The S-biotype of M. perniciosa infects solanaceous hosts, particularly pepper (Capsicum annuum) and tomato (Solanum lycopersicum). These species are much more amenable for performing studies of mechanisms underpinning host-pathogen interactions as compared to cacao. A phylogenetic analysis performed in this study demonstrated that S-biotype strains clustered with C-biotype strains, indicating that these biotypes are not genetically distinct. A comparative analysis demonstrated that disease progression in tomato infected with the S- biotype is similar to that described for cacao infected with the C- biotype. The major symptoms observed in both systems are swelling of the infected shoots and activation and proliferation of axillary meristems. Cellular changes observed in infected tissues correspond to an increase in cell size and numbers of xylem vessels and phloem parenchyma along the infected stem. Observations revealed that fungal colonization is biotrophic during the first phase of infection, with appearance of calcium oxalate crystals in close association with hyphal growth. In summary, despite different host specificity, both biotypes of M. perniciosa exhibit similar disease-related characteristics, indicating a degree of conservation of pathogenicity mechanisms between the two biotypes.     

Hydrosoluble phylloplane components of Theobroma cacao modulate the metabolism of Moniliophthora perniciosa spores during germination

The surface of plants forms a defense barrier that directly inhibits the first point of contact of microorganisms with the host. To understand this defense mechanism in Moniliophthora perniciosa interaction with Theobroma cacao cv Catongo, the aim of this study was to compare the changes in protein expression in basidiospores of the fungus M. perniciosa in response the leaf water washes (LWW) of two contrasting cocoa varieties for resistance to witches’ broom disease. A total of 8.1 × 10⁸ basidiospores were used for each treatment containing washed leaf material. Germinated basidiospores in the absence of LWW were used as control. The proteomic analysis was performed by the 2D-PAGE technique combined with mass spectrometry (MS). Protein extraction was based on the SDS-dense method followed by sonication for cell disruption and phenol extraction. Sixty-four proteins had accumulation of variation when compared to the control (no LWW). Proteins were identified associated with energy (ATP synthase) and protein (BiP) metabolism, whose accumulation was reduced by basidiospores germinated in leaf wash from Catongo cocoa. The reduction in ATP synthase of the basidiospores germinated the Catongo LWW suggests a shift from aerobic to fermentative metabolism of the fungus in response to components of the LWW. Furthermore, proteins involved in virulence were identified along with fungal resistance to polyketide cyclase, glycoside hydrolase, multidrug transporter protein (SFM) and proteins related to oxidative stress and fermentation, such as catalase A and alcohol dehydrogenase (ADH). The data showed an effect of cocoa phylloplane substances on the germination of fungal basidiospores through differences in protein expression patterns in the presence of LWW of the CCN51 and Catongo genotypes. These results may reveal mechanisms of resistance, host susceptibility and pathogen virulence.     

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.     

The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.     

Mycoplasmalike Organisms (MLOs) Associated With the Witches' Broom Disease of Poplar

The witches' broom disease has been recently observed on poplars in Paris and its suburbs. The incidence of the disease appeared to be considerably high along main roads. The electron microscopic examination of 350 nm thick sieve tube sections revealed the presence of wall-less mycoplasmalike organisms (MLOs) in diseased samples of Populus alba var. nivea Wesm. They could not be found in their healthy counterparts.     

抗枣疯病种质资源的筛选与应用

枣疯病是一种毁灭性的植原体病害。在全国范围内收集待鉴定抗枣疯病种质29份,采用在重病大树上高接待鉴定种质的高强度筛选方法,经过8年多的筛选和验证,从中筛选出4份抗性强且稳定的高抗单系,即骏枣单系、南京木枣单系、称砣枣单系、清徐圆枣单系。将骏枣高抗单系用于高接改造病树,已连续5年正常生长结果,未再次感染枣疯病。这些抗病单系既可直接作为抗病品种进行栽培,也可用于染病植株和感病品种的高接改造。     

A metaproteomic approach gives functional insights into anaerobic digestion

Aims: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. Methods and Results: The microbial communities from an anaerobic industrial‐like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum‐like (76%) micro‐organisms. Three hundred and eighty‐eight reproducible protein spots were obtained on 2‐D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO₂ and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro‐organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.     

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.     

Expression of the Theobroma cacao Bax‐inhibitor‐1 gene in tomato reduces infection by the hemibiotrophic pathogen Moniliophthora perniciosa

Programmed cell death (PCD) plays a key role in plant responses to pathogens, determining the success of infection depending on the pathogen lifestyle and on which participant of the interaction triggers cell death. The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao L. (cacao), a serious constraint for production in South America and the Caribbean. It has been hypothesized that M. perniciosa pathogenesis involves PCD, initially as a plant defence mechanism, which is diverted by the fungus to induce necrosis during the dikaryotic phase of the mycelia. Here, we evaluated whether the expression of a cacao anti‐apoptotic gene would affect the incidence and severity of M. perniciosa infection using the ‘Micro‐Tom’ (MT) tomato as a model. The cacao Bax‐inhibitor‐1 (TcBI‐1) gene, encoding a putative basal attenuator of PCD, was constitutively expressed in MT to evaluate function. Transformants expressing TcBI‐1, when treated with tunicamycin, an inducer of endoplasmic reticulum stress, showed a decrease in cell peroxidation. When the same transformants were inoculated with the necrotrophic fungal pathogens Sclerotinia sclerotiorum, Sclerotium rolfsii and Botrytis cinerea, a significant reduction in infection severity was observed, confirming TcBI‐1 function. After inoculation with M. perniciosa, TcBI‐1 transformant lines showed a significant reduction in disease incidence compared with MT. The overexpression of TcBI‐1 appears to affect the ability of germinating spores to penetrate susceptible tissues, restoring part of the non‐host resistance in MT against the S‐biotype of M. perniciosa.     

Integrative approach gives new insights into combined Cd/Cu exposure in tobacco

Heavy metals are generally known to induce oxidative stress, but are rarely strategically studied in an embracive manner, taking into account interplay between their various effects. Furthermore, although metals in the environment are present in mixtures and interact with each other, their combined effects to organisms have been much less studied in comparison to individual effects. Here, we present a complete comprehensive study of cadmium (Cd)/copper (Cu) oxidative stress interactions in Nicotiana tabacum seedlings and adult plants. Plants were treated with Cd (10 and 15 μM), Cu (2.5 and 5 μM) and their combinations; seedlings during 1 month period and adult plants during the period of 7 days. Metal accumulation measurements showed that Cd and Cu influence each other uptake, with Cu reducing Cd translocation to shoots. PCA analysis showed that MDA and carbonyls, biomarkers of oxidative stress, as well as ascorbate peroxidase activity, highly correlated across tissues and with Cd content. Majority of toxic effects were caused by Cd-alone, while addition of Cu often resulted in damage alleviation. However, mixture of high concentrations of both Cd and Cu induced most adverse effects. In conclusion, our results indicate that Cu in lower concentration has antagonistic effect to Cd toxicity, while in higher concentration these metals interact additively in tobacco.     

The Larix kaempferi genome reveals new insights into wood properties

Here, through single-molecule real-time sequencing,we present a high-quality genome sequence of the Japanese larch(Larix kaempferi), a conifer species with great value for wood production and ecological afforestation. The assembled genome is 10.97 Gb in size, harboring 45,828 protein-coding genes. Of the genome, 66.8% consists of repeat sequences, of which long terminal repeat retrotransposons are dominant and make up 69.86%. We find that tandem duplications have been responsible for the expansi...     

Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.