Five decades of research on phytoplasma-induced witches' broom diseases.

Phytoplasmas, prokaryotic wall-less microorganisms, are important pathogens of several plant species in most parts of the world. Phytoplasmas have been reported associated with various symptoms on hundreds of plant species. Witches' broom disease (WBD) is one of the most common disease symptoms, which is caused by phytoplasma strains belonging to different phytoplasma groups. Symptoms of the disease differ from one host to the other as well as from one phytoplasma strain to the other. However, WBD symptoms are usually characterized by the production of a large number of small leaves, accompanied in some host plants by the production of several branches/shoots. Phytoplasma strains belonging to more than 13 groups and 39 subgroups have been reported associated with WBD in more than 116 plant species. Most of the phytoplasma strains causing WBD symptoms in plant species belong to the 16SrII and 16SrI groups, mainly 16SrII-D and 16SrI-B subgroups. The current review provides information on the different types of phytoplasma strains associated with WBD symptoms in ornamental plants, medicinal plants, forest trees, weeds, vegetable crops, field crops, and fruit trees. Emphasis is on WBD on acid limes, almonds, peanuts, jujube, and cassava that have resulted in significant economic losses in different countries. Description of the symptoms, phytoplasma groups, and management options is also provided for some of the diseases.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches’ Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts

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.

The isolation and characterization of Taphrina betulina and other yeasts residing in the Betula pendula phylloplane.

The phylloplane is an important microbial habitat and a reservoir of organisms that affect plant health, both positively and negatively. Taphrina betulina is the causative agent of birch witches′ broom disease. Taphrina are dimorphic, invading theirs hosts in a filamentous form and residing in the host phyllosphere in their non-infectious yeast form. As such, they are widely accepted to be found a resident yeasts on their hosts, even on healthy tissues; however, there is little experimental data to support this. With the aim of exploring the local infection ecology of T. betulina, we have isolated yeasts from the phylloplane of birch, using three classes of samples; from infected symptom bearing leaves inside brooms, healthy leaves from branches away from brooms on broom bearing trees, and symptom-free leaves from symptom-free trees. Isolations yielded 224 yeast strains, representing 11 taxa, including T. betulina, which was the most common isolate and was found in all sample classes, including asymptomatic leaves. Genotyping with two genetic markers revealed genetic diversity among these T. betulina isolates, with seven distinct genotype differentiated by the markers used. Of the 57 T. betulina strains, 22 representative strains were selected for further studies and preliminarily characterized, revealing differences in size and the ability to produced compounds with activity to activate the signalling pathway for the plant hormone auxin.

Occurrence of a 16SrII-V Subgroup Phytoplasma Associated with Witches’-Broom Disease in Melochia corchorifolia in China

Melochia corchorifolia L. is a plant belonging to the family Sterculiaceae, extracts from this plant have been reported to inhibit melanogenesis (Yuan et al., 2020). During September to November 2020, the plants showing abnormal symptoms including witches’-broom, leaf chlorosis, leaflet and internode shortening (Fig.1), were found in Dingan county of Hainan province, China, with about 50% infection rates in the field. The disease symptoms were suspected to be caused by the phytoplasma, a plant pathogenic prokaryotes that could not be cultured in vitro. Aiming to confirm the pathogen causing the symptoms, total DNA of the symptomatic or asymptomatic Melochia corchorifolia samples were extracted by CTAB method (Doyle and Doyle, 1990) using 0.10 g fresh plant leaves using the rapid extraction kit for plant genomic DNA (CTAB Plant Genome DNA Rapid Extraction Kit, Aidlab Biotechnologies Co., Ltd, Beijing, China). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996) specific for phytoplasma 16S rRNA gene fragments. PCR products of phytoplasma 16S rRNA gene sequences were obtained from the ten symptomatic plant samples but not from the DNA of the asymptomatic plant samples. The PCR products were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the data were deposited in GenBank. The sequences of 16S rRNA gene fragments amplified from the DNA extracted from the disease plant samples were all identical, with a length of 1336 bp for the 16S rRNA (GenBank accession: MZ353520). Nucleotide Blast search based on the 16S rRNA gene fragment of the phytoplasma strain showed 100% sequence identities with that of 16SrII peanut witches’-broom group members, such as Cassava witches’-broom phytoplasma (KM280679), Cleome sp. phytoplasma (KM280677), Tephrosia purpurea witches’-broom phytoplasma (MW616560), Desmodium triflorum little leaf phytoplasma (MT452308) and Peanut witches’-broom phytoplasma (JX403944). Analysis of the 16S rRNA gene sequence of McWB-hnda strain by interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrII-V subgroup. The phytoplasma strain was named as Melochia corchorifolia witches’-broom (McWB) phytoplasma, McWB-hnda strain. Phylogenetic analysis performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016) indicated that the McWB-hnda phytoplasma strain was clustered into one clade with the phytoplasma strains of Tephrosia purpurea witches’-broom, Cleome sp., Peanut witches’-broom, Cassava witches’-broom and Desmodium triflorum little leaf with 97 % bootstrap value (Fig.2); McWB-hnda phytoplasma strain identified in the study and Melochia corchorifolia phyllody phytoplasma strain (KX150461) belonging to 16SrI-B subgroup previously identified in the Hainan Island of China by Chen et al. (2017) are in two independent clades(Fig.2). To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma associated with Melochia corchorifolia witches’-broom disease in Hainan Province, a tropical island of China. The phytoplasma strain identified in the study was relatively close to 16SrII peanut witches’-broom group phytoplasma strains associated with witches’-broom or little leaf diseases in the plants like Peanut, Tephrosia purpurea, Cassava and Desmodium triflorum. Our finding in the study indicated that Melochia corchorifolia may act as an alternative natural host not only for 16SrI-B subgroup phytoplasma but also for 16SrII-V subgroup phytoplasma, which would contribute to the spreading of the related phytoplasma diseases.

First report of ‘Candidatus Phytoplasma asteris’ associated with witches'-broom disease of Macadamia ternifolia in China

Macadamia nut (Macadamia ternifolia) was first introduced into China from Australia in 1910, and the main cultivation areas were Yunnan and Guangxi. It can be used as both a fruit and a therapeutic drug, with high economic value. In March 2021, it was observed that the M. ternifolia was showing witches'-broom, leaf yellowing and plexus bud in Dehong Prefecture, Yunnan Province, China. The terminal buds of infected plants were inhibited and the lateral buds are stimulated to germinate into twigs in advance. It was named the M. ternifolia witches'-broom disease, and was found in urban and rural areas of Mangshi, Lianghe, Yingjiang, Mangdong, Longchuan and Longling cities and counties. More than 40% of the plants were infected on the seven areas surveyed. The lateral stems from symptomatic and asymptomatic plants were cut to small pieces. The tissues were treated by fixation, dehydration and spraying-gold. And the tissues were observed under a scanning electron microscope (Hitachi S-3000N) (Pathan et al. 2010). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Symptomatic and asymptomatic plants were collected from seven areas, ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The total plants’ DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), and were stored at −20 °C in a refrigerator in the Key Laboratory of Forest Disaster Warning and Control at the Southwest Forestry University. The nested PCR was employed to amplify the 16S rRNA gene with the primers P1/P7 and R16F2/R16R2 (Lee et al. 1993; Schneider et al. 1993). PCR amplicon of 1.8 kb and 1.2 kb were amplified (GenBank accessions MW892818, MW892819, MW892820, MW892821). The direct PCR with primer pairs rp(I)F/ rp(I)R (Lee et al. 2003) specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb (GenBank accessions MZ442600, MZ442601, MZ442602, MZ442603). The fragment from 21 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. Interestingly, the phytoplasma/span>16S rRNA gene and rp gene was also amplified from the 4 samples of asymptomatic plant, we speculated that the latent infection and hidden symptoms existed in Macadamia nut (Moslemkhani and Sadeghi 2011). A BLAST analysis of the 16S rRNA sequences of MTWB phytoplasma showed that it has a 99% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99% identity with 'Salix tetradenia' witches'-broom phytoplasma (GenBank accession KC117314). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of MTWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628), with a pattern similarity coefficient of 0.99. The phytoplasma is identified as ‘Ca. Phytoplasma asteris’-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the MTWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In 2013, Macadamia nut showed leaf hardness phyllody and shoot proliferation caused by ‘Ca. Phytoplasma asteris’ in Artemisa, Cuba. The concern is that, the macadamia phytoplasma is closely related to the subgroup 16SrI-F, and it is significantly different from the Chinese strains (Pérez-López et al. 2013). In addition, the MTWB phytoplasma was graft-transmitted from infected to healthy plants in nursery conditions (Akhtar et al. 2009; Ikten et al. 2014). And the grafted plants were positive for the phytoplasma in the nested PCR assays. It is noteworthy that the plants were seriously damaged by aphid and it was speculated that the insects of Homoptera caused the spread of the disease by sucking plant sap, thus the aphids that transmits MTWB in China must be determined to control the M. ternifolia witches'-broom disease. To the best of our knowledge, Macadamia nut is a new host plant of ‘Ca. Phytoplasma asteris’ in China. The newly emerged disease is a threat to Macadamia nut.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle

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.