Validation of PCR diagnostic assays for detection and identification of all Ralstonia solanacearum sequevars causing Moko disease in banana.

Moko disease in banana is a bacterial wilt caused by strains within Ralstonia solanacearum sensu stricto. The disease is endemic to Central and South America but has spread to the Philippines and peninsular Malaysia. Detecting new incursions early in Moko-free banana production regions is of utmost importance for containment and eradication, as Moko management significantly increases costs of banana production. Molecular studies have supported the classification of R. solanacearum sensu stricto into phylotypes IIA, IIB and IIC, each comprising of various sequevars based on nucleotide divergence of a partial sequence within the endoglucanase gene. Moko disease in banana is caused by strains classified as sequevars 6, 24, 41, and 53 within phylotype IIA, and sequevars 3, 4, and 25 within phylotype IIB. To ensure accurate diagnostic assays are available to detect all Moko sequevars, we systematically validated previously published assays for Moko diagnostics. To be able to identify all sequevars, including the latest described sequevars, namely IIB-25, IIA-41, and IIA-53, we developed and validated two novel assays using genome-wide association studies on over 100 genomes of R. solanacearum sensu stricto. Validations using 196 bacterial isolates confirmed that a previous multiplex PCR based assay targeting sequevars IIB-3, IIB-4, IIA-6 and IIA-24 and our two novel assays targeting sequevars IIB-25, IIA-41 and IIA-53 were specific, reproducible, and accurate for Moko diagnostics.

Selection for Phytophthora Root Rot Resistance in Chickpea Crosses Affects Yield Potential of Chickpea × Cicer echinospermum Backcross Derivatives.

Phytophthora root rot (PRR) of chickpea (Cicer arietinum) caused by Phytophthora medicaginis is an important disease. Partial resistance to PRR is sourced from Cicer echinospermum. In this study, we evaluated if lines with low levels of PRR foliage symptoms in two contrasting recombinant inbred line (RIL) populations parented by chickpea cultivars (Yorker and Rupali) and 04067-81-2-1-1 (C. echinospermum, interspecific breeding line) had a significant drag on yield parameters. For the Yorker × 04067-81-2-1-1 population with the highest level of PRR resistance, in the absence of PRR, low foliage symptom RIL had significantly later flowering and podding, lower grain yields, and lighter seed and shorter plant phenotypes than high foliage symptom RIL. A quantitative trait locus analysis identified significant QTL for flowering, height, 100-seed weight, and yield, and there was a significantly higher frequency of alleles for the negative agronomic traits (i.e., drag) from the 04067-81-2-1-1 parent in low foliage symptom RIL than in high foliage symptom RIL. For the Rupali × 04067-81-2-1-1 population with lower levels of PRR resistance, in the absence of PRR, low foliage symptom RIL had significantly lighter seed and shorter plants than high foliage symptom RIL. Significant QTL were detected, the majority were for the timing of flowering and podding (n = 18), others were for plant height, yield, and 100-seed weight. For this second population, the frequency of alleles for the negative agronomic traits from the 04067-81-2-1-1 parent did not differ between low and high foliage symptom RIL. The 100 seed weight of RIL under moderate PRR disease pressure showed some promise as a yield component trait to identify phenotypes with both high levels of PRR resistance and grain yield potential for further seed number evaluations. We identified that large population sizes are required to enable selection among chickpea × C. echinospermum crosses for high levels of PRR resistance without a significant drag on yield.

Segmental duplications drive the evolution of accessory regions in a major crop pathogen.

Many pathogens evolved compartmentalized genomes with conserved core and variable accessory regions (ARs) that carry effector genes mediating virulence. The fungal plant pathogen Fusarium oxysporum has such ARs, often spanning entire chromosomes. The presence of specific ARs influences the host range, and horizontal transfer of ARs can modify the pathogenicity of the receiving strain. However, how these ARs evolve in strains that infect the same host remains largely unknown. We defined the pan-genome of 69 diverse F. oxysporum strains that cause Fusarium wilt of banana, a significant constraint to global banana production, and analyzed the diversity and evolution of the ARs. Accessory regions in F. oxysporum strains infecting the same banana cultivar are highly diverse, and we could not identify any shared genomic regions and in planta-induced effectors. We demonstrate that segmental duplications drive the evolution of ARs. Furthermore, we show that recent segmental duplications specifically in accessory chromosomes cause the expansion of ARs in F. oxysporum. Taken together, we conclude that extensive recent duplications drive the evolution of ARs in F. oxysporum, which contribute to the evolution of virulence.

Domestication through clandestine cultivation constrained genetic diversity in magic mushrooms relative to naturalized populations.

Fungi that are edible or fermentative were domesticated through selective cultivation of their desired traits. Domestication is often associated with inbreeding or selfing, which may fix traits other than those under selection, and causes an overall decrease in heterozygosity. A hallucinogenic mushroom, Psilocybe cubensis, was domesticated from its niche in livestock dung for production of psilocybin. It has caused accidental poisonings since the 1940s in Australia, which is a population hypothesized to be introduced from an unknown center of origin. We sequenced genomes of 38 isolates from Australia and compared them with 86 genomes of commercially available cultivars to determine (1) whether P. cubensis was introduced to Australia, and (2) how domestication has impacted commercial cultivars. Our analyses of genome-wide SNPs and single-copy orthologs showed that the Australian population is naturalized, having recovered its effective population size after a bottleneck when it was introduced, and it has maintained relatively high genetic diversity based on measures of nucleotide and allelic diversity. In contrast, domesticated cultivars generally have low effective population sizes and hallmarks of selfing and clonal propagation, including low genetic diversity, low heterozygosity, high linkage disequilibrium, and low allelic diversity of mating-compatibility genes. Analyses of kinship show that most cultivars are founded from related populations. Alleles in the psilocybin gene cluster are identical across most cultivars of P. cubensis with low diversity across coding sequence; however, unique allelic diversity in Australia and some cultivars may translate to differences in biosynthesis of psilocybin and its analogs.

Inoculum production of Phytophthora medicaginis can be used to screen for partial resistance in chickpea genotypes.

Phytophthora root rot caused by Phytophthora medicaginis is an important disease of chickpeas (Cicer arietinum) in Australia with limited management options, increasing reliance on breeding for improved levels of genetic resistance. Resistance based on chickpea-Cicer echinospermum crosses is partial with a quantitative genetic basis provided by C. echinospermum and some disease tolerance traits originating from C. arietinum germplasm. Partial resistance is hypothesised to reduce pathogen proliferation, while tolerant germplasm may contribute some fitness traits, such as an ability to maintain yield despite pathogen proliferation. To test these hypotheses, we used P. medicaginis DNA concentrations in the soil as a parameter for pathogen proliferation and disease assessments on lines of two recombinant inbred populations of chickpea-C. echinospermum crosses to compare the reactions of selected recombinant inbred lines and parents. Our results showed reduced inoculum production in a C. echinospermum backcross parent relative to the C. arietinum variety Yorker. Recombinant inbred lines with consistently low levels of foliage symptoms had significantly lower levels of soil inoculum compared to lines with high levels of visible foliage symptoms. In a separate experiment, a set of superior recombinant inbred lines with consistently low levels of foliage symptoms was tested for soil inoculum reactions relative to control normalised yield loss. The in-crop P. medicaginis soil inoculum concentrations across genotypes were significantly and positively related to yield loss, indicating a partial resistance-tolerance spectrum. Disease incidence and the rankings for in-crop soil inoculum were correlated strongly to yield loss. These results indicate that soil inoculum reactions may be useful to identify genotypes with high levels of partial resistance.

Genome sequencing progenies of magic mushrooms (Psilocybe subaeruginosa) identifies tetrapolar mating and gene duplications in the psilocybin pathway.

Knowledge of breeding systems and genetic diversity is critical to select and combine desired traits that advance new cultivars in agriculture and horticulture. Mushrooms that produce psilocybin, magic mushrooms, may potentially be used in therapeutic and wellness industries, and stand to benefit from genetic improvement. We studied haploid siblings of Psilocybe subaeruginosa to resolve the genetics behind mating compatibility and advance knowledge of breeding. Our results show that mating in P. subaeruginosa is tetrapolar, with compatibility controlled at a homeodomain locus with one copy each of HD1 and HD2, and a pheromone/receptor locus with four homologs of the receptor gene STE3. An additional two pheromone/receptor loci homologous to STE3 do not appear to regulate mating compatibility. Alleles in the psilocybin gene cluster did not vary among the five siblings and were likely homozygous in the parent. Psilocybe subaeruginosa and its relatives have three copies of PsiH genes but their impact on production of psilocybin and its analogues is unknown. Genetic improvement in Psilocybe will require access to genetic diversity from the centre of origin of different species, identification of genes behind traits, and strategies to avoid inbreeding depression.

Transmission of Blood Disease in Banana.

Banana Blood disease is a bacterial wilt caused by Ralstonia syzygii subsp. celebesensis and is an economically important disease in Indonesia and Malaysia. Transmission of this pathogen is hypothesized to occur through insects mechanically transferring bacteria from diseased to healthy banana inflorescences and other pathways involving pruning tools, water movement, and root-to-root contact. This study demonstrates that the ooze from the infected male bell and the sap from various symptomatic plant parts are infective, and the cut surfaces of a bunch peduncle, petiole, corm, pseudostem, and the rachis act as infection courts for R. syzygii subsp. celebesensis. In addition, evidence is provided that R. syzygii subsp. celebesensis is highly tool transmissible, that the bacterium can be transferred from the roots of a diseased plant to the roots of a healthy plant and transferred from the mother plant to the sucker. We provide evidence that local dispersal of Blood disease occurs predominantly through mechanical transmission by insects, birds, bats, or human activities from diseased to healthy banana plants and that long-distance dispersal occurs through the movement of contaminated planting material. Disease management strategies to prevent crop losses associated with this emerging disease are discussed based on our findings.

Susceptibility of the Banana Inflorescence to Blood Disease.

The bacterium Ralstonia syzygii subsp. celebesensis causes Blood disease of banana, a vascular wilt of economic significance in Indonesia and Malaysia. Blood disease has expanded its geographic range in the last 20 years and is an emerging threat to Southeast Asian banana production. Many aspects of the disease cycle and biology are not well understood, including the ability of different parts of the female and male inflorescence of banana to act as infection courts. This study confirms that the banana varieties of Cavendish, and Kepok 'Kuning' are susceptible to Blood disease and that an inoculum concentration of 102 CFU/ml of R. syzygii subsp. celebesensis is adequate to initiate disease after pseudostem inoculation. Data show that infection occurs through both the male and female parts of a banana inflorescence and the rachis when snapped to remove the male bell. The infection courts are the female flowers, the male bell bract scar, the male bell flower cushion, the snapped rachis, and deflowered fingers. The location of these infection courts concurs with the dye studies demonstrating that dye externally applied to these plants parts enters the plant vascular system. Thus, the hypothesis is supported that infection of R. syzygii subsp. celebesensis occurs through open xylem vessels of the male and female parts of the banana inflorescence.

Diagnostics of Banana Blood Disease.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt disease that causes major yield losses of banana in Indonesia and peninsular Malaysia. The disease has significantly increased its geographic distribution in the past decade. Diagnostic methods are an important component of disease management in vegetatively propagated crops such as banana to constrain incursions of plant pathogens. Therefore, the objectives of this study were (i) to design and rigorously validate a novel banana Blood disease (BBD) real-time PCR assay with a high level of specificity and sensitivity of detection and (ii) to validate published PCR-based diagnostic methods targeting the intergenic region in the megaplasmid ("121 assay" with primer set 121) or the phage tail protein-coding sequence in the bacterial chromosome ("Kubota assay" and "BDB2400 assay" with primer set BDB2400). Assay validation included 339 samples (174 Blood disease bacteria, 51 bacteria associated with banana plants, 51 members of the Ralstonia solanacearum species complex, and 63 samples from symptomatic and healthy plant material). Validation parameters were analytical specificity (inclusivity and exclusivity), selectivity, limit of detection, accuracy, and ruggedness. The 121 assay and our newly developed BBD real-time PCR assay detected all R. syzygii subsp. celebesensis strains with no cross-specificity during validation. Two different PCR assays using the primer set BDB2400 lacked specificity and selectivity. This study reveals that our novel BBD real-time PCR assay and the conventional PCR 121 assay are reliable methods for Blood disease diagnostics, as they comply with all tested validation parameters.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

The Vulnerability of Bananas to Globally Emerging Disease Threats.

Banana cultivation has increased significantly over the last century to meet the growing demand for this popular fruit. Originating in Southeast Asia, bananas are now produced in >135 different countries in tropical and subtropical regions of the world. Most of this expansion of production is based on a single banana variety, Cavendish, which makes up almost all the export trade grown in large-scale monocultures and a large part of the local trade and represents >40% of all bananas grown globally. Over the last century several major diseases of the banana have emerged and widely expanded their geographic ranges. Cultivars within the Cavendish variety are highly susceptible to these diseases, including yellow Sigatoka, black leaf streak, Eumusae leaf spot, freckle, Fusarium wilt tropical race 4, banana bunchy top, and the bacterial wilts Moko, Xanthomonas wilt, and banana Blood disease. This review graphically illustrates the emergence and rapid intercontinental spread of these diseases and discusses several major disease epidemics in bananas. Evidently, the large-scale monoculture based on the single variety Cavendish has resulted in an extreme level of genetic vulnerability. The resistance to diversification in the Cavendish production chain and the lack of investment in genetics and plant breeding in the recent past means that currently limited genetic solutions are available to replace the Cavendish banana with a set of market acceptable resistant varieties from a range of different genetic backgrounds.

Geographic Expansion of Banana Blood Disease in Southeast Asia.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt causing significant crop losses in Indonesia and Malaysia. Disease symptoms include wilting of the plant and red-brown vascular staining, internal rot, and discoloration of green banana fruit. There is no known varietal resistance to this disease in the Musa genus, although variation in susceptibility has been observed, with the popular Indonesian cooking banana variety Kepok being highly susceptible. This study established the current geographic distribution of Blood disease in Indonesia and confirmed the pathogenicity of isolates by Koch's postulates. The long-distance distribution of the disease followed an arbitrary pattern indicative of human-assisted movement of infected banana materials. In contrast, local or short-distance spread radiated from a single infection source, indicative of dispersal by insects and possibly contaminated tools, water, or soil. The rapid expansion of its geographical range makes Blood disease an emerging threat to banana production in Southeast Asia and beyond.

Inoculum Dynamics and Infection of Citrus Fruit by Phyllosticta citricarpa.

Citrus black spot, caused by Phyllosticta citricarpa, is characterized by fruit blemishes and premature fruit drop, resulting in significant economic losses in summer rainfall areas. The pathogen forms both conidia and ascospores during its life cycle. However, the occurrence of these spores and their contributions to infection of fruit in field conditions are not well understood. Our research using direct leaf litter monitoring and volumetric spore trapping in Queensland orchards revealed that pseudothecia and ascospores in leaf litter as well as trapped ascospores had low abundance, while pycnidia and conidia were highly abundant. Both P. citricarpa and endophytic Phyllosticta spp. were identified, with P. citricarpa being dominant. In replicated field trials, we determined that infection of Imperial mandarin fruit by P. citricarpa occurred from fruit set until week 20 of fruit development, with the key infection events taking place between weeks 4 and 16 in Queensland subtropical conditions. These results demonstrate that protecting fruit during weeks 4 to 16 significantly reduced P. citricarpa infection. We found no significant correlation between the disease incidence in fruit and P. citricarpa conidial abundance in leaf litter or ascospore abundance measured by volumetric spore trapping. Therefore, it is suggested that inoculum sources in the tree canopy other than those detected by spore trapping and direct leaf litter monitoring may play a major role in the epidemiology of citrus black spot. Improved knowledge regarding epidemiology of P. citricarpa and an understanding of propagules causing infection may aid in development of more effective disease management strategies.

Transcriptomic data of the Musa balbisiana cultivar Kepok inoculated with Ralstonia syzigii subsp. celebesensis and Ralstonia solanacearum.

The increasing production of banana is hampered by the spread of banana plant diseases, one of which is caused by a group of bacteria, including those of causing wilt diseases. In Indonesia, blood disease is one of the important banana wilt diseases since loss due to the infection can reach up to 50%. There are numerous publications on the pathogen identification causing banana blood disease based on the molecular approach, however to date, no detailed molecular data are available for the interaction of banana host plant against the pathogen. Here, we present three raw data sets of the total RNA-seq from the inoculated Musa balbisiana cutivar kepok (ABB genome) inoculated with Ralstonia syzigii subsp. celebesensis, Ralstonia solanacearum and mock. The data provide essential knowledge for differentiating the banana response against pathogen, reveal pathogenesis-related genes and gene functions in the plant system, and research development to design blood disease-resistance of banana as one of the management strategies. Raw reads of RNA-seq data can be found in NCBI's Sequence Read Archive (SRA) database with the accession number of SRR10547839 (RSC), SRR10547840 (RS), and SRR10547841 (Mock).

Molecular Diagnostics of Banana Fusarium Wilt Targeting Secreted-in-Xylem Genes.

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Fungal clones win the battle, but recombination wins the war.

Clonal reproduction is common in fungi and fungal-like organisms during epidemics and invasion events. The success of clonal fungi shaped systems for their classification and some pathogens are tacitly treated as asexual. We argue that genetic recombination driven by sexual reproduction must be a starting hypothesis when dealing with fungi for two reasons: (1) Clones eventually crash because they lack adaptability; and (2) fungi find a way to exchange genetic material through recombination, whether sexual, parasexual, or hybridisation. Successful clones may prevail over space and time, but they are the product of recombination and the next successful clone will inevitably appear. Fungal pathogen populations are dynamic rather than static, and they need genetic recombination to adapt to a changing environment.

New Geographical Insights of the Latest Expansion of Fusarium oxysporum f.sp. cubense Tropical Race 4 Into the Greater Mekong Subregion.

Banana is the most popular and most exported fruit and also a major food crop for millions of people around the world. Despite its importance and the presence of serious disease threats, research into this crop is limited. One of those is Panama disease or Fusarium wilt. In the previous century Fusarium wilt wiped out the "Gros Michel" based banana industry in Central America. The epidemic was eventually quenched by planting "Cavendish" bananas. However, 50 years ago the disease recurred, but now on "Cavendish" bananas. Since then the disease has spread across South-East Asia, to the Middle-East and the Indian subcontinent and leaped into Africa. Here, we report the presence of Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4) in "Cavendish" plantations in Laos, Myanmar, and Vietnam. A combination of classical morphology, DNA sequencing, and phenotyping assays revealed a very close relationship between the Foc TR4 strains in the entire Greater Mekong Subregion (GMS), which is increasingly prone to intensive banana production. Analyses of single-nucleotide polymorphisms enabled us to initiate a phylogeography of Foc TR4 across three geographical areas-GMS, Indian subcontinent, and the Middle East revealing three distinct Foc TR4 sub-lineages. Collectively, our data place these new incursions in a broader agroecological context and underscore the need for awareness campaigns and the implementation of validated quarantine measures to prevent further international dissemination of Foc TR4.

Pathogenicity of Phyllosticta citricarpa Ascospores on Citrus spp.

Citrus black spot, caused by Phyllosticta citricarpa, is one of the most important fungal diseases in many citrus-growing regions with hot and humid summers. Ascospores and conidia are known to contribute to epidemic development of the disease. However, pathogenicity testing has never been done for pure ascospores produced from fully characterized P. citricarpa isolates, due to the inability to induce the sexual state in vitro. Recently, an in vitro mating technique was developed to readily produce pure P. citricarpa ascospores for use in host inoculation studies. To test the pathogenicity of P. citricarpa ascospores, we inoculated Troyer citrange leaves and Murcott tangor fruit with ascospores produced in vitro from characterized P. citricarpa isolates. Typical symptoms of citrus black spot occurred. Recovery of P. citricarpa isolates from symptomatic lesions and their characterization using genetic markers enabled us to identify recombinant genotypes among the isolates recovered from ascospore inoculations and, as such, fulfill Koch's postulates for ascospores. We have also identified Troyer citrange seedlings as a potential model system for citrus black spot inoculation studies, because it allows typical symptoms of citrus black spot to be expressed with a much shorter latent period than on fruit. This will facilitate future studies of epidemiological aspects of P. citricarpa ascospores relative to conidia and improve our understanding of the citrus black spot pathosystem. The susceptibility of Troyer citrange seedlings will also facilitate experimenting with disease management methods, aimed at reducing the impact of citrus black spot.