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Β-glucosidase (BGLs) act synergistically with endoglucanases and exoglucanases and then are of great interest for biomass conversion into bioethanol. Thus, the aim of the current study is to produce a recombinant β-glycosidase from Moniliophtora perniciosa expressed in Escherichia coli cells. Enzyme coding sequence expression was confirmed through Sanger sequencing after using wheat bran (WB) and carboxymethylcellulose (CMC) as fungal growth media. Synthetic gene betaglyc-GH1 with optimized codons for E. coli expression was cloned in pET-28a. β-glucosidase recombinant (GH1chimera) was purified using a nickel column and its identity was confirmed through mass spectrometry. The recombinant enzyme presented an apparent molecular mass of 53.23 kDa on SDS-PAGE. Recombinant β-glucosidase has shown hydrolytic activity using p-nitrophenyl-β-D-glycopyranoside (pNPG) as substrate and maximum activity at pH 4.6 and 65 °C. Thus, the results indicate that the application of the GH1chimera in the hydrolysis of lignocellulosic materials to obtain glucose monomers can be efficient. [ABSTRACT FROM AUTHOR]

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches' broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao-M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, ß-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase (P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease. [ABSTRACT FROM AUTHOR]

BACKGROUND: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. RESULTS: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. CONCLUSIONS: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Bamboo is used for making structures, furniture, handicrafts, and ropes, as well as a source of food in the Philippines. One of the emerging diseases of bamboo in the country is the bamboo witches' broom (BWB), which has been occasionally noted in three genera of bamboo - including Dendrocalamus, Gigantochloa, and Schizostachyum from various provinces in the Philippines (Ilocos Norte, Laguna, Batangas, Quezon, Agusan del Sur, Bukidnon, and South Cotabato) since the 1990s. However, studies and information about BWB in the country remain lacking and largely unexplored. In this study, we report a similar disease affecting Dendrocalamus and Gigantochloa bamboo species from Bohol and Davao and - for the first time - in Dendrocalamus merrillianus ("bayog") and Bambusa spinosa ("kawayan-tinik") from Isabela and Nueva Vizcaya. As a result of the surveys conducted from 1999-2019, the disease is now identified in six species across four genera of bamboo - namely, Gigantochloa spp. (G. levis and G. atter), Dendrocalamus spp. (D. asper and D. merrillianus), Schizostachyum lumampao, and Bambusa spinosa recorded in 11 provinces in the Philippines. The BWB symptoms include clustering of leaves forming a rosette-like structure, leaf proliferation, excessive limb formation from a single node, and shortening of internodes. Nested PCR using the universal primers P1/P7 and R16MF2/R1 targeting the phytoplasma 16S ribosomal RNA gene revealed positive amplification in five symptomatic BWB samples from Isabela, Philippines. Subsequent sequencing (~ 1.3kbp) and phylogenetic analysis using the representative BWB isolates from Isabela revealed > 98.65% genetic similarity and clustering to Candidatus Phytoplasma luffae, which belongs to the 16SrVIII group (Loofah Witches' Broom Group). This paper determined the distribution of BWB in different species of bamboo in the Philippines, as well as the association of 'Ca. Phytoplasma luffae'-related strain (16SrVIII) to BWB. [ABSTRACT FROM AUTHOR]

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins with pivotal roles in plant defense mechanisms. In this study, various bioinformatics and RNA-seq methods were used to analyze the biotic and abiotic stress responses of the TLP family in Phyllostachys edulis. Overall, 81 TLP genes were identified in P. edulis; 166 TLPs from four plant species were divided into three groups and ten subclasses, with genetic covariance observed between these species. Subcellular localization in silico studies indicated that TLPs were primarily distributed in the extracellular. Analysis of the upstream sequences of TLPs demonstrated the presence of cis-acting elements related to disease defense, environmental stress, and hormonal responses. Multiple sequence alignment demonstrated that most TLPs possessed five conserved REDDD amino acid sequences with only a few amino acid residue differences. RNA-seq analysis of P. edulis responses to Aciculosporium take, the pathogenic fungus that causes witches' broom disease, showed that P. edulis TLPs (PeTLPs) were expressed in different organs, with the highest expression in buds. PeTLPs responded to both abscisic acid and salicylic acid stress. These PeTLP expression patterns were consistent with their gene and protein structures. Collectively, our findings provide a basis for further comprehensive analyses of the genes related to witches' broom in P. edulis. [ABSTRACT FROM AUTHOR]

A set of diseases caused by fungi and oomycetes are responsible for large losses in annual world cocoa production. Managing the impact caused by these diseases is very complex because a common solution has yet to be found for different pathogens. In this context, the systematic knowledge of Theobroma cacao L. pathogens' molecular characteristics may help researchers understand the possibilities and limitations of cocoa disease management strategies. This work systematically organized and summarized the main findings of omics studies of T. cacao eukaryotic pathogens, focusing on the plant–pathogen interaction and production dynamics. Using the PRISMA protocol and a semiautomated process, we selected papers from the Scopus and Web of Science databases and collected data from the selected papers. From the initial 3169 studies, 149 were selected. The first author's affiliations were mostly from two countries, Brazil (55%) and the USA (22%). The most frequent genera were Moniliophthora (105 studies), Phytophthora (59 studies) and Ceratocystis (13 studies). The systematic review database includes papers reporting the whole-genome sequence from six cocoa pathogens and evidence of some necrosis-inducing-like proteins, which are common in T. cacao pathogen genomes. This review contributes to the knowledge about T. cacao diseases, providing an integrated discussion of T. cacao pathogens' molecular characteristics, common mechanisms of pathogenicity and how this knowledge is produced worldwide. [ABSTRACT FROM AUTHOR]

Abstract Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

A set of diseases caused by fungi and oomycetes are responsible for large losses in annual world cocoa production. Managing the impact caused by these diseases is very complex because a common solution has yet to be found for different pathogens. In this context, the systematic knowledge of Theobroma cacao L. pathogens’ molecular characteristics may help researchers understand the possibilities and limitations of cocoa disease management strategies. This work systematically organized and summarized the main findings of omics studies of T. cacao eukaryotic pathogens, focusing on the plant–pathogen interaction and production dynamics. Using the PRISMA protocol and a semiautomated process, we selected papers from the Scopus and Web of Science databases and collected data from the selected papers. From the initial 3169 studies, 149 were selected. The first author’s affiliations were mostly from two countries, Brazil (55%) and the USA (22%). The most frequent genera were Moniliophthora (105 studies), Phytophthora (59 studies) and Ceratocystis (13 studies). The systematic review database includes papers reporting the whole-genome sequence from six cocoa pathogens and evidence of some necrosis-inducing-like proteins, which are common in T. cacao pathogen genomes. This review contributes to the knowledge about T. cacao diseases, providing an integrated discussion of T. cacao pathogens’ molecular characteristics, common mechanisms of pathogenicity and how this knowledge is produced worldwide.

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection. [ABSTRACT FROM AUTHOR]

The witches' broom disease of cacao, caused by the fungus Moniliophthora perniciosa, is among the main diseases of cacao (Theobroma cacao L.), one of the most important cultures in the tropics, for which chemical control is not very effective. We suggested that the epicuticular wax layer of cacao pods might have antifungal activity against Moniliophthora perniciosa. The objective of this article was to establish a protocol for extraction of the epicuticular layer of cacao pods and establish a bioassay to evaluate the in vitro antifungal activity of these epicuticular extracts on germination of M. perniciosa. First, we standardized the extraction protocol, and extracts of the epicuticular layer from standard, susceptible, and resistant cacao clones to WBDC were obtained. Then, we used an in vitro agar bioassay to evaluate the antifungal activity of the extracts against M. perniciosa basidiospores. The inhibition of the germination was highest (87.2%) for spores treated with the extracts from the resistant genotype. Treatment with extracts from ICS1 (susceptible genotype) did not significantly influence spore germination. We successfully extracted T. cacao wax and suggested that the epicuticular layer of cacao genotypes has antifungal molecules with inhibitory activity against the germination of M. perniciosa basidiospores in vitro. [ABSTRACT FROM AUTHOR]

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches' Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça , and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa , originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts' antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides. [ABSTRACT FROM AUTHOR]

Witches' broom of birch (Betula spp.) caused by Taphrina betulina is an understudied disease that causes the formation of woody tumours, from which ectopic axillary buds and branches grow to form a broom‐like structure. We have addressed two aspects of this disease using naturally infected mature trees in the field. Broom symptoms offer a convenient means of scoring susceptibility in the field. Variation in broom symptom presentation suggests possible variation in resistance against witches' broom disease. We tracked the local distribution of susceptible individuals among 721 trees at 159 independent sites. The analysis supports the hypothesis that there was genetic resistance segregating in these birch populations. Anatomical changes in broom symptom bearing branches of European silver birch (Betula pendula) were also addressed by comparing sections of tissues from three locations in the same branch, which were normal, swollen in infected tissue adjacent to a tumour, and inside a tumour. Examination of tumours revealed disorganized and swollen xylem, expanded secondary phloem and expanded periderm. Swollen tissues newly infected from spreading disease adjacent to tumours exhibited enhanced growth only in secondary phloem and the periderm, which also exhibited distortions. This finding suggests that tumour formation and possibly pathogen colonization may initiate in these tissues. [ABSTRACT FROM AUTHOR]

Summary: Moniliophthoraperniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanumlycopersicum) model system Micro‐Tom (MT) by the Solanaceae (S)‐biotype of Moniliophthoraperniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism.Inoculation of an MT‐transgenic line that overexpresses the Arabidopsis CYTOKININOXIDASE‐2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA‐sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin‐responsive marker genes when symptoms were conspicuous.The expression of an MoniliophthoraperniciosatRNA‐ISOPENTENYL‐TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown invitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans‐zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR‐991 and PI55 decreased symptoms.Moniliophthoraperniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus. [ABSTRACT FROM AUTHOR]

Loofah [Luffa acutangula (L.) Roxb.] is grown in some regions of Asia, Africa and the Americas. This vegetable is well known for its high nutritional and medicinal value. Loofah plants showing typical symptoms of phytoplasma infection, such as witches' broom, fruit deformation, flower proliferation and phyllody were observed in a commercial field in Sinaloa, Mexico, in January 2015. Nineteen symptomatic loofah plants and seven asymptomatic weeds (Helianthus annuus, Sarcostemma sp., Chenopodium sp., Amaranthus palmeri, Rumex sp., Ambrosia sp., and Raphanus sp.) growing nearby were collected and analyzed by phytoplasma-specific nested PCR. Phytoplasmas were detected in 79% of symptomatic loofah plants and in two of the seven weeds. Conventional and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rRNA-encoding gene sequences indicated the presence of strains of the 16SrIII and 16SrI phytoplasma groups in loofah, which occurred as both single and mixed infections. The phylogenetic analysis confirmed four new phytoplasma strains associated with loofah diseases, two 'Candidatus Phytoplasma asteris'-related strains (16SrI-AG and 16SrI-AH), and two 'Candidatus Phytoplasma pruni'-related strains (16SrIII-AA, and 16SrIII-AB). Furthermore, phytoplasmas belonging to groups 16SrI and 16SrIII also were identified in the weeds H. annuus and A. palmeri, respectively; 16SrIII-AC represents a new phytoplasma subgroup described in A. palmeri in Sinaloa. To our knowledge, this is the first report of loofah disease associated with phytoplasmas belonging to the 16SrI and 16SrIII groups in Mexico. [ABSTRACT FROM AUTHOR]

Sweet orange (Citrus sinensis) and Orlando tangelo (C. paradisi × C. reticulata) trees showing witches'-broom symptoms were observed during 2018 in southern Kerman province, Iran. Detection of phytoplasmas in the symptomatic trees was confirmed by nested PCR assays using the phytoplasma specific primers. Sequence analysis indicated that the phytoplasmas associated with the witches'-broom affected trees belong to the 16SrII-C subgroup phytoplasma. This is the first report of a 16SrII-C phytoplasma associated with witches'-broom symptoms of Sweet orange and Orlando tangelo trees in the world. [ABSTRACT FROM AUTHOR]

Witches' broom caused by Moniliophthora perniciosa is the main disease of cacao (Theobroma cacao) in Brazil. The fungus is known to occur on other host families and these populations have been addressed in the literature as biotypes: C (Malvaceae); H (Malpighiaceae); L (Bignoniaceae) and S (Solanaceae). No complete elucidation of the phylogenetic relationships of isolates obtained from this disparate host range appears in the literature. One member of H (ex Heteropterys acutifolia) has been described as a distinct species. But should other biotypes be also recognized as distinct taxa? In the present study, a survey yielding 24 isolates of M. perniciosa from ten hosts and covering a wide range of geographic regions in Brazil was undertaken. These isolates were compared with those from T. cacao using three DNA regions for the phylogenetic analyses: ITS, LSU and RPB1. Morphology was also examined. All isolates in this study were found to belong to M. perniciosa , including the population from H. acutifolia , formerly treated as Moniliophthora brasiliensis but reduced here to a synonym of M. perniciosa. This species ranged from pathogenic to a previously unreported occurrence as a non-pathogenic endophyte in the Atlantic rainforest tree Allophylus edulis (Sapindaceae). M. perniciosa was recorded on a range of solanaceous hosts (16 species) over a wide variety of ecosystems. The ecological and evolutionary significance of these novel findings are discussed. [ABSTRACT FROM AUTHOR]

The cultivars BRS Careca, BRS Fartura, BRS Duquesa, BRS Curinga, and BRS Golias of Theobroma grandiflorum are characterized by high levels of fruit production and diverse sources of resistance to witches' broom disease. These cultivars are recommended for replacing the tree canopy of low yielding genotypes or forming new orchards. [ABSTRACT FROM AUTHOR]

The cupuassu tree (Theobroma grandiflorum) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu- M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin. • TgPRs are chitinases and osmotine with distinct 3D structures. • TgPRs interacted with chitin and chitosan, having higher affinity energy to chitosan. • The osmotine TgPR5 may act as chitinase/chitosanase. • TgPRs were highly expressed in resistant cupuassu infected by Moniliphthora perniciosa. • TgPR may participate to the cupuassu innate immune system in the early interaction stage. [ABSTRACT FROM AUTHOR]

Witches' broom disease of Citrus aurantifolia (Christm.) Swingle, caused by 'Candidatus Phytoplasma aurantifolia', is a big threat to Citrus aurantifolia production in South of Iran. Accumulation of defense-related enzymes is defense reactions against pathogens in infected plant tissues. Herein, the effect of ethyl acetate extract of Camellia sinensis (L.) Kuntze was investigated on induced resistance against phytoplasma in the leaves of Citrus aurantifolia. The leaves were pretreated with different concentration of the extract, and then study on the defense enzymes and phenolic compounds was done. The results indicated that the pre-treatment of the leaves with low concentration of Camellia sinensis extract induced resistance through the accumulation of various phenolic compounds and phytoalexins, as well as the activation of peroxidases (POD), catalase (CAT) and key enzymes in phenylpropanoid pathways i.e., phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and stilbene synthase (STS). Thus, applying low concentration of Camellia sinensis extract induces resistance against disease and can be a novel strategy in plant disease management. [ABSTRACT FROM AUTHOR]

Proteins from the glutathione peroxidase (GPX) family, such as GPX4 or PHGPX in animals, are extensively studied for their antioxidant functions and apoptosis inhibition. GPXs can be selenium-independent or selenium-dependent, with selenium acting as a potential cofactor for GPX activity. However, the relationship of plant GPXs to these functions remains unclear. Recent research indicated an upregulation of Theobroma cacao phospholipid hydroperoxide glutathione peroxidase gene (Tc PHGPX) expression during early witches' broom disease stages, suggesting the use of antioxidant mechanisms as a plant defense strategy to reduce disease progression. Witches' broom disease, caused by the hemibiotrophic fungus Moniliophthora perniciosa , induces cell death through elicitors like Mp NEP2 in advanced infection stages. In this context, in silico and in vitro analyses of Tc PHGPX's physicochemical and functional characteristics may elucidate its antioxidant potential and effects against cell death, enhancing understanding of plant GPXs and informing strategies to control witches' broom disease. Results indicated Tc PHGPX interaction with selenium compounds, mainly sodium selenite, but without improving the protein function. Protein-protein interaction network suggested cacao GPXs association with glutathione and thioredoxin metabolism, engaging in pathways like signaling, peroxide detection for ABA pathway components, and anthocyanin transport. Tests on tobacco cells revealed that Tc PHGPX reduced cell death, associated with decreased membrane damage and H 2 O 2 production induced by Mp NEP2. This study is the first functional analysis of Tc PHGPX, contributing to knowledge about plant GPXs and supporting studies for witches' broom disease control. • This is the first functional analysis of PHGPX from Theobroma cacao. • Tc PHGPX can interact with both organic and inorganic selenium compounds. • Tc PHGPX structure can withstand temperatures above ambient and up to 52 °C. • Tc PHGPX reduced membrane damage, peroxide accumulation, and necrosis-type PCD. • Tc PHGPX activity is independent of sodium selenite. [ABSTRACT FROM AUTHOR]

The article discusses a 2021 report on the increased infection of Artemisia sieberi plants in Iran by a 16Srll-C phytoplasma strain linked to Artemisia sieberi witches'-broom disease. It states that infected plants had little leaf, yellowing, internode shortening, and witches'-broom symptoms, and that the Circulifer hoematoceps leafhopper can transmit the strain. Also noted is the first occurrence of A. sieberi as new host of the 16Srll-C phytoplasma subgroup in Iran in the report.

The article discusses a 2021 report on the infection of cactus plants in Turkey by Witches' broom disease symptoms linked to Candidatus Phytoplasma aurantifolia. It states that symptoms of yellowing, shoot proliferations, leaf discoloration and redness appeared in infected cactus, and that similar-diseased plants have been reported in Mexico, Lebanon, and China. Also noted is the increasing Ca. P. aurantifolia strain in Turkey that can infect plants in nature and in shops.

Plant diseases caused by fungal pathogens are responsible for major crop losses worldwide, with a significant socio-economic impact on the life of millions of people who depend on agriculture-exclusive economy. This is the case of the Witches’ Broom Disease (WBD) affecting cacao plant and fruit in South and Central America. The severity and extent of this disease is prospected to impact the growing global chocolate market in a few decades. WBD is caused by the basidiomycete fungus Moniliophthora perniciosa. The methods used to contain the fungus mainly rely on chemical fungicides, such as copper-based compounds or azoles. Not only are these highly ineffective, but also their utilization is increasingly restricted by the cacao industry, in part because it promotes fungal resistance, in part related to consumers’ health concerns and environmental awareness. Therefore, the disease is being currently tentatively controlled through phytosanitary pruning, although the full removal of infected plant material is impossible and the fungus maintains persistent inoculum in the soil, or using an endophytic fungal parasite of Moniliophthora perniciosa which production is not sustainable. The growth of Moniliophthora perniciosa was reported as being antagonized in vitro by some yeasts, which suggests that they could be used as biological control agents, suppressing the fungus multiplication and containing its spread. Concurrently, some yeast-based products are used in the protection of fruits from postharvest fungal spoilage, and the extension of diverse food products shelf-life. These successful applications suggest that yeasts can be regarded a serious alternative also in the pre-harvest management of WBD and other fungal plant diseases. Yeasts’ GRAS (Generally Recognized as Safe) nature adds to their appropriateness for field application, not raising major ecological concerns as do the present more aggressive approaches. Importantly, mitigating WBD, in a sustainable manner, would predictably have a high socioeconomic impact, contributing to diminish poverty in the cacao-producing rural communities severely affected by the disease. This review discusses the importance/advantages and the challenges that such a strategy would have for WBD containment, and presents the available information on the molecular and cellular mechanisms underlying fungi antagonism by yeasts.

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa , the causal agent of witches' broom disease (WBD) in Theobroma cacao , initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao - M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, β-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase ( P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease., Competing Interests: The author(s) declare no conflict of interest.

Moniliophthora perniciosa is the causal agent of the witches' broom disease (WBD) in cacao but the morphological and molecular diversity of M. perniciosa in Ecuador—the top fine flavor cacao producer worldwide—are poorly understood and the pathogen's sensitivity to compost teas is unknown. A total of 90 isolates of M. perniciosa were obtained from symptomatic samples of cocoa branches and pods collected from four cocoa-producing regions of Ecuador. Growth of each isolate was assed and fitted to a Gompertz model, and the molecular variability was evaluated by sequencing the ITS1, 5.8s, and ITS2 rDNA regions as well as by inter-retrotransposon amplified polymorphism (IRAP), retrotransposon microsatellite amplified polymorphism (REMAP), and simple sequence repeats (SSR) analyses. Sensitivity of M. perniciosa to a compost tea was evaluated at concentrations of 0.5, 1, 1.5, 2, 2.75, and 3.5% in PDA. Results showed morphological and growth rate homogeneity across isolates. Mycelial growth fitted to Gompertz model gave similar parameter estimates for isolates from the different sampling sites. No molecular variability was observed based on ITS and SSR analysis but IRAP and REMAP tests showed high polymorphism. The total genetic diversity based on IRAP and REMAP tests was 0.28 and the genetic diversity within population was 0.24, with Nei's diversity, Shannon's Information, and percent polymorphism values of 0.315, 0.48, and 95.24, respectively for the whole sampled population; isolates from Los Rios province being the most polymorphic. Compost tea sensitivity analysis performed on 85 of the isolates showed a significant inhibitory effect at concentrations of 2.75% or above but concentrations below 1.5% significantly enhanced the growth of most isolates of the pathogen. Results confirmed the high genetic diversity of M. perniciosa and provided insights into growth rate and compost tea sensitivity of the pathogen in Ecuador. [ABSTRACT FROM AUTHOR]

Plant diseases caused by fungal pathogens are responsible for major crop losses worldwide, with a significant socio-economic impact on the life of millions of people who depend on agriculture-exclusive economy. This is the case of the Witches' Broom Disease (WBD) affecting cacao plant and fruit in South and Central America. The severity and extent of this disease is prospected to impact the growing global chocolate market in a few decades. WBD is caused by the basidiomycete fungus Moniliophthora perniciosa. The methods used to contain the fungus mainly rely on chemical fungicides, such as copper-based compounds or azoles. Not only are these highly ineffective, but also their utilization is increasingly restricted by the cacao industry, in part because it promotes fungal resistance, in part related to consumers' health concerns and environmental awareness. Therefore, the disease is being currently tentatively controlled through phytosanitary pruning, although the full removal of infected plant material is impossible and the fungus maintains persistent inoculum in the soil, or using an endophytic fungal parasite of Moniliophthora perniciosa which production is not sustainable. The growth of Moniliophthora perniciosa was reported as being antagonized in vitro by some yeasts, which suggests that they could be used as biological control agents, suppressing the fungus multiplication and containing its spread. Concurrently, some yeast-based products are used in the protection of fruits from postharvest fungal spoilage, and the extension of diverse food products shelf-life. These successful applications suggest that yeasts can be regarded a serious alternative also in the pre-harvest management of WBD and other fungal plant diseases. Yeasts' GRAS (Generally Recognized as Safe) nature adds to their appropriateness for field application, not raising major ecological concerns as do the present more aggressive approaches. Importantly, mitigating WBD, in a sustainable manner, would predictably have a high socioeconomic impact, contributing to diminish poverty in the cacao-producing rural communities severely affected by the disease. This review discusses the importance/advantages and the challenges that such a strategy would have for WBD containment, and presents the available information on the molecular and cellular mechanisms underlying fungi antagonism by yeasts. [ABSTRACT FROM AUTHOR]

Highlights • Mp-AOX diminishes hydrogen peroxide generation by mitochondria. • Mp-AOX has antioxidant function. • Mp-AOX expression decreases growth ratio and biomass of S. cerevisiae. • S. cerevisiae expressing Mp-AOX as high-throughput platform to inhibitor screening. Abstract The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition. [ABSTRACT FROM AUTHOR]

Background: Moniliophthora perniciosa (Stahel) Aime & Phillips‐Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane‐bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. Results: We present the first study aimed at developing novel fungal AOX inhibitors. N‐Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j‐41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j‐41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure‐activity relationships analyses. Conclusion: We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry The enzyme alternative oxidase is a target for fungicide development. We designed, synthesized, screened and validated a novel compound with antifungal activity against phytopathogens that attack cocoa and other crops. [ABSTRACT FROM AUTHOR]

Witches’ broom disease has led to major losses in lime and alfalfa production in Oman. This paper identifies bioclimatic variables that contribute to the prediction of distribution of witches’ broom disease in current and future climatic scenarios. It also explores the expansion, reduction, or shift in the climatic niche of the distribution of the disease across the different geographical areas of the entire country (309,501 km²). The maximum entropy model (MaxEnt) and geographical information system were used to investigate the potential suitability of habitats for the phytoplasma disease. This study used current (1970–2000) and future projected climatic scenarios (2021–2040, 2041–2060, 2061–2080, and 2081–2100) to model the distribution of phytoplasma for lime trees and alfalfa in Oman. Bioclimatic variables were downloaded from WorldClim with ± 60 occurrence points for lime trees and alfalfa. The area under the curve (AUC) was used to evaluate the model’s performance. Quantitatively, the results showed that the mean of the AUC values for lime (16SrII-B) and alfalfa (16SrII-D) future distribution for the periods of 2021–2040, 2041–2060, 2061–2080, and 2081–2100 were rated as “excellent”, with the values for the specified time periods being 0.859, 0.900, 0.931, and 0.913 for 16SrII-B; and 0.826, 0.837, 08.58, and 0.894 for 16SrII-D respectively. In addition, this study identified the hotspots and proportions of the areas that are vulnerable under the projected climate-change scenarios. The area of current (2021–2040) highly suitable distribution within the entire country for 16SrII-D was 19474.2 km2 (7.1%), while for 16SrII-B, an area of 8835 km2 (3.2%) was also highly suitable for the disease distribution. The proportions of these suitable areas are very significant from the available arable land standpoint. Therefore, the results from this study will be of immense benefit and will also bring significant contributions in mapping the areas of witches’ broom diseases in Oman. The results will equally aid the development of new strategies and the formulation of agricultural policies and practices in controlling the spread of the disease across Oman.

Cacao (Theobroma cacao) is a globally important crop, and its yield is severely restricted by disease. Two of the most damaging diseases, witches’ broom disease (WBD) and frosty pod rot disease (FPRD), are caused by a pair of related fungi: Moniliophthora perniciosa and Moniliophthora roreri, respectively. Resistant cultivars are the most effective long-term strategy to address Moniliophthora diseases, but efficiently generating resistant and productive new cultivars will require robust methods for screening germplasm before field testing. Marker-assisted selection (MAS) and genomic selection (GS) provide two potential avenues for predicting the performance of new genotypes, potentially increasing the selection gain per unit time. To test the effectiveness of these two approaches, we performed a genome-wide association study (GWAS) and GS on three related populations of cacao in Ecuador genotyped with a 15K single nucleotide polymorphism (SNP) microarray for three measures of WBD infection (vegetative broom, cushion broom, and chirimoya pod), one of FPRD (monilia pod) and two productivity traits (total fresh weight of pods and % healthy pods produced). GWAS yielded several SNPs associated with disease resistance in each population, but none were significantly correlated with the same trait in other populations. Genomic selection, using one population as a training set to estimate the phenotypes of the remaining two (composed of different families), varied among traits, from a mean prediction accuracy of 0.46 (vegetative broom) to 0.15 (monilia pod), and varied between training populations. Simulations demonstrated that selecting seedlings using GWAS markers alone generates no improvement over selecting at random, but that GS improves the selection process significantly. Our results suggest that the GWAS markers discovered here are not sufficiently predictive across diverse germplasm to be useful for MAS, but that using all markers in a GS framework holds substantial promise in accelerating disease-resistance in cacao.

Summary: Phytoplasmas are insect‐transmitted phytopathogenic bacteria, which secrete effector proteins that are often responsible for altering the plant morphology and behaviours of their vectors. Phytoplasma multifunctional effector proteins TENGU and SAP11 induce typical witches' broom symptoms, but their mode of action remains unknown. Previously, we have identified a SAP11‐like effector from wheat blue dwarf phytoplasma, SWP1, which induces witches' broom symptoms in Nicotiana benthamiana. In this study, we observed that SWP1‐expressing transgenic Arabidopsis thaliana plants showed typical witches' broom symptoms. On overexpression of SWP1 truncation mutants in N. benthamiana, we identified that the coiled‐coil domain and nuclear localization were responsible for the induction of witches' broom symptoms. In addition, using yeast two‐hybrid and bimolecular fluorescence complementation assays, we demonstrated that SWP1 interacts with A. thaliana transcription factor TCP18 (BRC1), the key negative regulator of branching signals in various plant species. Moreover, in planta co‐expression analysis showed that SWP1 promotes the degradation of BRC1 via a proteasome system. These findings suggest that the phytoplasma effector SWP1 induces witches' broom symptoms through targeting of BRC1 and promoting its degradation. [ABSTRACT FROM AUTHOR]

Phytoplasmas are insect-transmitted, phloem-inhabiting, cell wall-less prokaryotes that cause numerous diseases in plants. A survey carried out during 2012-2013 in peach and nectarine orchards in three Northern provinces including Mazandaran (Sari, Neka, Babol, Behshahr and Babolsar), Golestan (Gorgan, kordkooy and Aliabad) and West Azerbaijan (Urmia, Khoy, Salmas, Oshnavieh and Makou) in Iran. A total of 103 Samples showing leaf roll, yellowing, purple colored of edges, leaf stunting and weak shoot growth symptoms were collected and tested by polymerase chain reaction (PCR) and Nested -PCR using primer pairs P1/P7, P1/Tint, PAF/PAR, R16F2n/R2, fU5/rU3 and NPA1F/NPA1R. The amplicons were purified, sequenced and the nucleotide sequences were analyzed by virtual restriction fragment length polymorphism (RFLP). The phytoplasmas associated with the peach and nectarine yellow leaf roll (PYLR) disease were identified as members of 16SrXII (Stolbur group) and 16SrIX (pigeon pea) groups and were placed in a unique subgroup close to 16SrXII-A (Candidatus Phytoplasma solani) and another one in 16SrIX-C (Candidatus Phytoplasma phoenciium) subgroups, respectively in constructed phylogenetic trees. The present study is the first report of a new subgroup in 16SrXII and first report of 16SrIX-C subgroup phytoplasma associated with peach and nectarine from North of Iran. [ABSTRACT FROM AUTHOR]

The article discusses the development of transgenic plants producing plantibodies against Candidatus Phytoplasma aurantifolia. It states that the witches' broom of lime is a disease caused by Ca. Phytoplasma aurantifolia which is the most significant global threat to the production of lime trees (Citrus aurantifolia). It also states that disease management strategies have shown little success and new approaches based on genetic engineering need to be considered like the produced plantibody.

Background: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. Results: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. Conclusions: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity. [ABSTRACT FROM AUTHOR]

Summary: Taxonomy: Moniliophthora roreri (Cif.) H.C. Evans et al. ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology: Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45–90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa: Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches’ broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance: Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries. Disease management: The disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available. [ABSTRACT FROM AUTHOR]

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Abstract The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao ( Theobroma cacao ) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response. Highlights • We report the cloning, expression, and purification of kinase domains of two PR-1 Receptor-Like Kinases (PR-1Rk). • Recombinant PR-1RK were expressed in E. coli BL21(DE3)-R3-pRARE2 and BL21(DE3)-R3-lambda-PPase. • Co-expression of lambda-PPase were critical for the production of the recombinant kinase domains in soluble form. [ABSTRACT FROM AUTHOR]

The identification of effectors from pathogenic microbes is one of the most important subjects for elucidating infection mechanisms. Wheat blue dwarf (WBD) phytoplasma causes dwarfism, witches' broom, and yellow leaf tips in wheat plants, resulting in severe yield loss in northwestern China. In this study, 37 candidate effector proteins were transiently expressed in Nicotiana benthamiana. Plants expressing the SAP11‐like protein SWP1 exhibited typical witches' broom. Interestingly, another protein, SWP11, induced both cell death and defence responses, including H2O2 accumulation and callose deposition. Analysis by qRT‐PCR was used to show that a marker gene of the hypersensitive response, HIN1, and three pathogenesis‐related genes, PR1, PR2 and PR3, were significantly up‐regulated in leaves of N. benthamiana expressing SWP11. In addition, SWP12 and SWP21 (TENGU‐like) were shown to suppress SWP11‐, BAX‐, and/or INF1‐induced cell death. These results indicated that SWP21 has a distinct role in virulence compared with TENGU and that WBD phytoplasma possesses effectors that target plant proliferation and defence responses. The ability of these effectors to trigger or suppress plant immunity provides new insights into the phytoplasma–plant interaction. [ABSTRACT FROM AUTHOR]

Cacao (Theobroma cacao) is a globally important crop, and its yield is severely restricted by disease. Two of the most damaging diseases, witches' broom disease (WBD) and frosty pod rot disease (FPRD), are caused by a pair of related fungi: Moniliophthora perniciosa and Moniliophthora roreri, respectively. Resistant cultivars are the most effective long-term strategy to address Moniliophthora diseases, but efficiently generating resistant and productive new cultivars will require robust methods for screening germplasm before field testing. Marker-assisted selection (MAS) and genomic selection (GS) provide two potential avenues for predicting the performance of new genotypes, potentially increasing the selection gain per unit time. To test the effectiveness of these two approaches, we performed a genome-wide association study (GWAS) and GS on three related populations of cacao in Ecuador genotyped with a 15K single nucleotide polymorphism (SNP) microarray for three measures of WBD infection (vegetative broom, cushion broom, and chirimoya pod), one of FPRD (monilia pod) and two productivity traits (total fresh weight of pods and % healthy pods produced). GWAS yielded several SNPs associated with disease resistance in each population, but none were significantly correlated with the same trait in other populations. Genomic selection, using one population as a training set to estimate the phenotypes of the remaining two (composed of different families), varied among traits, from a mean prediction accuracy of 0.46 (vegetative broom) to 0.15 (monilia pod), and varied between training populations. Simulations demonstrated that selecting seedlings using GWAS markers alone generates no improvement over selecting at random, but that GS improves the selection process significantly. Our results suggest that the GWAS markers discovered here are not sufficiently predictive across diverse germplasm to be useful for MAS, but that using all markers in a GS framework holds substantial promise in accelerating disease-resistance in cacao. [ABSTRACT FROM AUTHOR]

Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species. [ABSTRACT FROM AUTHOR]

Symptoms of stunting (shortening of internodes), twisting and flat stem (the fasciation of a stem), discoloration of petals, deformed flowers, and witches' broom were recorded on an ornamental plant, plumed cockscomb ( Celosia argentea L., fam: Amaranthaceae). The survey conducted at Indian Agricultural Research Institute (IARI) campus, New Delhi and Karnal region, Haryana, India, during September 2014 to March 2015 revealed disease incidence of 40 and 10%, respectively. The 16S rRNA gene sequence comparison and phylogenetic relationships of Celosia phytoplasma strains under study confirmed that they were associated with two different phytoplasma groups (' Candidatus Phytoplasma australasia' and ' Ca. P. asteris'). Virtual RFLP analysis of 16S rRNA gene sequences allowed further classification of the Celosia phytoplasma strains into the 16SrI-B and 16SrII-D subgroups. Notably, the detection of ' Ca. P. asteris' phytoplasma was reported in seeds of C. argentea by nested PCR assays; however, no evidence of phytoplasma presence was detected in seedlings raised from these seeds. This observation is the first record of the association of 16SrI-B and 16SrII-D subgroups of phytoplasmas with flat stem and witches' broom disease of C. argentea anywhere from the world. [ABSTRACT FROM AUTHOR]

Witches' broom disease ( WBD), caused by ' Candidatus Phytoplasma aurantifolia', is a serious disease of acid lime ( Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ' Candidatus Phytoplasma aurantifolia' was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II-B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800-801 000 copies) in leaves developing WBD symptoms compared to 2-268 copies in symptomless leaves from the same trees and 8-874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression. [ABSTRACT FROM AUTHOR]

Nine geographically diverse Moniliophthora perniciosa (witches' broom disease pathogen) isolates were cultured in vitro . They included six C-biotypes differing in virulence on cacao ( Theobroma cacao ), two S-biotypes (solanaceous hosts), and an L-biotype (liana hosts). Mycelial growth rates and morphologies differed considerably, but no characters were observed to correlate with virulence or biotype. In plant inoculations using basidiospores, one C-biotype caused symptoms on tomato (an S-biotype host), adding to evidence of limited host adaptation in these biotypes. Mycelial proteomes were analysed by two-dimensional gel electrophoresis (2-DE), and 619 gel spots were indexed on all replicate gels of at least one strain. Multivariate analysis of gel spots discriminated the L-biotype, but not the S-biotypes, from the remaining strains. The proteomic similarity of the S- and C-biotypes is consistent with their reported lack of phylogenetic distinction. Sequences from tandem mass spectrometry of tryptic peptides from major 2-DE spots were matched with Moniliophthora genome and transcript sequences on NCBI and WBD Transcriptome Atlas databases. Protein-spot identifications indicated that M. perniciosa saprotrophic mycelial proteomes expressed functions potentially connected with a ‘virulence life-style’, including peroxiredoxin, heat-shock proteins, nitrilase, formate dehydrogenase, a prominent complement of aldo-keto reductases, mannitol-1-phosphate dehydrogenase, and central metabolism enzymes with proposed pathogenesis functions. [ABSTRACT FROM AUTHOR]

Recently, Russian olive trees showing witches'-broom and little leaf symptoms have been widely observed in northwestern and central Iran. Polymerase chain reaction (PCR) and nested PCR assays using phytoplasma universal primer pairs confirmed phytoplasma symptomatic infection of trees. Sequence analyses showed that 'Candidatus Phytoplasma asteris' was the causal agent of the disease in these regions. However, RFLP results using restriction enzymes HpaII, EcoRI, HinfI and AluI indicated that the collected isolates in these regions are genetically different. In addition, leafhopper Macropsis infuscata was recognized as a possible insect vector of the disease for the first time. [ABSTRACT FROM AUTHOR]

Background: Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches' broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. Results: A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. Conclusions: In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease. [ABSTRACT FROM AUTHOR]

Moniliophthora perniciosa is the causal agent of witches' broom in Theobroma cacao (cacao). Three biotypes of M. perniciosa are recognized, differing in host specificity, with two causing symptoms on cacao or Solanaceae species (C- and S-biotypes), and the third found growing endophytically on lianas (L-biotype). The objectives of this study were to clarify the genetic relationship between the three biotypes, and to identify those regions in the Brazilian Amazon with the greatest genetic diversity for the C-biotype. Phylogenetic reconstruction based on the rRNA ITS regions showed that the C- and S-biotypes formed a well-supported clade separated from the L-biotype. Analysis of 131 isolates genotyped at 11 microsatellite loci found that S- and especially L-biotypes showed a higher genetic diversity. A significant spatial genetic structure was detected for the C-biotype populations in Amazonia for up to 137 km, suggesting 'isolation by distance' mode of dispersal. However, in regions containing extensive cacao plantings, C-biotype populations were essentially 'clonal', as evidenced by high frequency of repeated multilocus genotypes. Among the Amazonian C-biotype populations, Acre and West Amazon displayed the largest genotypic diversity and might be part of the centre of diversity of the fungus. The pathogen dispersal may have followed the direction of river flow downstream from Acre, Rondônia and West Amazon eastward to the rest of the Amazon valley, where cacao is not endemic. The Bahia population exhibited the lowest genotypic diversity, but high allele richness, suggesting multiple invasions, with origin assigned to Rondônia and West Amazon, possibly through isolates from the Lower Amazon population. [ABSTRACT FROM AUTHOR]