Vegetative Compatibility Research Articles

Genetic variation, vegetative compatibility, and aggressiveness diversity of Diplodia bulgarica isolates from apple orchards in West Azarbaijan province of Iran

AbstractThis study investigated inter‐simple sequence repeat (ISSR), vegetative compatibility, and aggressiveness diversity in 101 isolates of Diplodia bulgarica recovered from apple trees displaying symptoms of canker and decline in West Azarbaijan province of Iran. Marker analyses revealed high within population diversity, low genetic differentiation, high gene flow, and sharing of multilocus genotypes (MLGs) among geographic populations. Moreover, clustering and multivariate analyses identified two highly differentiated genetic clusters with limited admixture between them. These findings may suggest that the pathogen has been introduced from two genetically divergent sources and has been moved within the region through infected materials. The large number of MLGs, low clonal fraction, and absence of a widely distributed dominant genotype may explain the occurrence of recombination in this pathogen. However, significant linkage disequilibrium in the populations and limited admixture between genetic clusters may indicate the rare occurrence of recombination in D. bulgarica populations in West Azarbaijan, and that the pathogen has not been in the province long enough to reach equilibrium. Vegetative compatibility analyses revealed the occurrence of anastomosis between nonself pairings and high vegetative compatibility group diversity within populations. All studied MLGs produced necrotic lesions on detached shoots of Red Delicious apple but differed in their aggressiveness levels. Our results provide new insights into genetic and phenotypic variation of D. bulgarica that can assist in developing management strategies. Our findings also highlight the vital need for quarantine measures and the production of healthy plant materials to prevent the introduction and spread of the pathogen in Iran.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

Key Insights and Research Prospects at the Dawn of the Population Genomics Era for Verticillium dahliae.

The genomics era has ushered in exciting possibilities to examine the genetic bases that undergird the characteristic features of Verticillium dahliae and other plant pathogens. In this review, we provide historical perspectives on some of the salient biological characteristics of V. dahliae, including its morphology, microsclerotia formation, host range, disease symptoms, vascular niche, reproduction, and population structure. The kaleidoscopic population structure of this pathogen is summarized, including different races of the pathogen, defoliating and nondefoliating phenotypes, vegetative compatibility groupings, and clonal populations. Where possible, we place the characteristic differences in the context of comparative and functional genomics analyses that have offered insights into population divergence within V. dahliae and the related species.Current challenges are highlighted along with some suggested future population genomics studies that will contribute to advancing our understanding of the population divergence in V. dahliae.

Secreted in Xylem Genes: Drivers of Host Adaptation in Fusarium oxysporum.

Fusarium oxysporum (Fo) is a notorious pathogen that significantly contributes to yield losses in crops of high economic status. It is responsible for vascular wilt characterized by the browning of conductive tissue, wilting, and plant death. Individual strains of Fo are host specific (formae speciales), and approximately, 150 forms have been documented so far. The pathogen secretes small effector proteins in the xylem, termed as Secreted in Xylem (Six), that contribute to its virulence. Most of these proteins contain cysteine residues in even numbers. These proteins are encoded by SIX genes that reside on mobile pathogenicity chromosomes. So far, 14 proteins have been reported. However, formae speciales vary in SIX protein profile and their respective gene sequence. Thus, SIX genes have been employed as ideal markers for pathogen identification. Acquisition of SIX-encoding mobile pathogenicity chromosomes by non-pathogenic lines, through horizontal transfer, results in the evolution of new virulent lines. Recently, some SIX genes present on these pathogenicity chromosomes have been shown to be involved in defining variation in host specificity among formae speciales. Along these lines, the review entails the variability (formae speciales, races, and vegetative compatibility groups) and evolutionary relationships among members of F. oxysporum species complex (FOSC). It provides updated information on the diversity, structure, regulation, and (a)virulence functions of SIX genes. The improved understanding of roles of SIX in variability and virulence of Fo has significant implication in establishment of molecular framework and techniques for disease management. Finally, the review identifies the gaps in current knowledge and provides insights into potential research landscapes that can be explored to strengthen the understanding of functions of SIX genes.

Mycelial compatibility, anastomosis, and nucleus numbers of eight Mexican Hirsutella citriformis strains isolated from Diaphorina citri.

BackgroundAmong entomopathogenic fungi, H. citriformis has been recognized as potential biocontrol agent against the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Nevertheless, this fungus is poorly characterized. Previous molecular studies have shown high sequence similarities among strains, but significant differences in Diaphorina citri virulence.ObjectiveThe aim of the present study was to determine mycelial compatibility and anastomosis, and nucleus numbers in mycelium and conidia of eight H. citriformis strains isolated from mycosed D. citri adults collected from several Mexican states.MethodsMycelial compatibility and anastomosis evaluation was performed after pairing strains, leading to 36 confrontations, and cultured in chlorate minimum medium to obtain mutants for vegetative compatibility group.ResultsHypha or conidia nuclei were visualized with safranin-O and 3% KOH, and 0.05% trypan blue–lactophenol solution. H. citriformis strains showed compatibly and anastomosis events after confrontation. In addition, they showed one nucleus per conidium and mycelium section. It was not possible to obtain H. citriformis nit mutants from the chlorate concentrations tested.ConclusionsTo date, this is the first report demonstrating mycelial compatibility, anastomosis occurrence, and hyphae and conidia nuclei number among H. citriformis strains.

Take-All Disease: New Insights into an Important Wheat Root Pathogen.

Take-all disease, caused by the fungal root pathogen Gaeumannomyces tritici, is considered to be the most important root disease of wheat worldwide. Here we review the advances in take-all research over the last 15 years, focusing on the identification of new sources of genetic resistance in wheat relatives and the role of the microbiome in disease development. We also highlight recent breakthroughs in the molecular interactions between G. tritici and wheat, including genome and transcriptome analyses. These new findings will aid the development of novel control strategies against take-all disease. In light of this growing understanding, the G. tritici-wheat interaction could provide a model study system for root-infecting fungal pathogens of cereals.

Phylogenetic Analysis, Vegetative Compatibility, Virulence, and Fungal Filtrates of Leaf Curl Pathogen Colletotrichum fioriniae from Celery.

Leaf curl of celery, caused by Colletotrichum acutatum sensu lato, has been reported in the United States. A multilocus phylogenetic analysis with three genes was conducted with a collection of isolates from celery (n = 23) and noncelery (n = 29) hosts to evaluate their taxonomic position within C. acutatum sensu lato. The three DNA regions used for phylogenetic analysis included the introns of the glutamine synthase GS and glyceraldehyde-3-phosphate dehydrogenase GPDH genes, and the partial sequence of the histone3 his3 gene. Moreover, celery and noncelery isolates were evaluated for vegetative compatibility and pathogenicity on celery. Culture filtrates from celery and noncelery isolates were also evaluated for their ability to reproduce leaf curl symptoms. A total of 23 celery isolates were evaluated based on phylogenetic analysis, which showed that all celery isolates were closely related and belonged to the newly described species C. fioriniae. The celery isolates were grouped into six vegetative compatibility groups, indicating that the population was not clonal. Isolates of C. fioriniae from celery (22 of 23) and other hosts (26 of 29) caused leaf curl symptoms. Isolates of C. acutatum, C. nymphaeae, and C. godetiae were pathogenic but did not cause leaf curl symptoms. Isolates of C. lupini, C. johnstonii, and C. gloeosporioides were not pathogenic on celery. In addition, cell-free fungal culture filtrates caused leaf curl symptoms on celery, indicating that certain isolates produce a metabolite that can cause leaf curl symptoms on celery, possibly indole acetic acid.

Genetic variability and vegetative compatibility in Aspergillus niger isolated from various food substances in Benin City Nigeria

In this investigation, Aspergillus niger isolated from eight food substances, have been classified based on the absence of heterokaryon formation. The size of their sporangia were differentiated, the wild and mutant strains were subjected to vegetative compatibility tests in order to group them into different vegetative compatibility groups (VCGs) which include VCG-1, VCG-2, VCG-3 and VCG-4. The strains were further tested for the possible formation of a stable heterokaryon using nit mutants generated on potato dextrose agar (PDA) containing 2.5% chlorate (KClO3), represented as PDC. Based on the vegetative compatibility groups, nit mutants were paired on a minimal medium (MM) for complementation test. Interestingly, there was compatibility with mycelia showing anastomoses but without the formation of heterokaryon. The vegetative compatibility groups suggested four genotypes and polymorphism in the het loci. A population study for detailed genotyping is suggested in order to unravel the genetic recombination in A. niger.

Temporal and Spatial Genetic Population Structure of Cryphonectria parasitica and Its Associated Hypovirus Across an Invasive Range of Chestnut Blight in Europe.

Chestnut blight has spread throughout Europe since the introduction of its causal agent, Cryphonectria parasitica, >70 years ago. In our study, we analyzed the diversity of vegetative compatibility (vc) and microsatellite genotypes of C. parasitica, as well as sequence diversity of Cryphonectria hypovirus 1 (CHV1) in six populations from Switzerland, Croatia, and North Macedonia. Resampling of local populations that were already investigated more than a decade ago allowed us to analyze the spatial and temporal population structure across an invasive range of the pathogen in Europe. Regardless of which genetic marker was used, the >60-year-old Swiss and Croatian populations had high population diversity, whereas more recent North Macedonian populations were mostly clonal. These diversity differences between the investigated populations remained stable over time. A high diversity of CHV1 was observed in all three countries, with North Macedonian strains forming a separate cluster from strains obtained in other countries. No correlation between vc diversity and CHV1 prevalence was observed, suggesting a well-established and maintained natural hypovirulence in all countries, further corroborated by an observed increase in genetic diversity of Croatian C. parasitica populations over time, without collapse of CHV1 prevalence.

The Verticillium wilt problem in Australian cotton

Verticillium dahliae is a soil-borne phytopathogen and the causal agent of Verticillium wilt. It affects many agriculturally important crops around the world, including cotton. In Australia, the billion-dollar cotton industry is increasingly impacted by Verticillium wilt. Internationally it has been reported that the defoliating V. dahliae Vegetative Compatibility Group (VCG) 1A causes severe damage to cotton. In Australia however, the non-defoliating VCG2A is causing more severe damage to crops in fields than the defoliating VCG1A. This review examines the current research to understand the Australian V. dahliae situation, including current classification systems, genetic analyses and management strategies. It appears that virulence cannot be defined solely by VCG in Australian Verticillium dahliae isolates causing disease in cotton, and that the industry must continually adapt their practices in order to keep the disease under control.

Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.

Genetic Differentiation of Verticillium dahliae Populations Recovered from Symptomatic and Asymptomatic Hosts.

Verticillium dahliae is a soilborne fungal pathogen affecting many economically important crops that can also infect weeds and rotational crops with no apparent disease symptoms. The main research goal was to test the hypothesis that V. dahliae populations recovered from asymptomatic rotational crops and weed species are evolutionarily and genetically distinct from symptomatic hosts. We collected V. dahliae isolates from symptomatic and asymptomatic hosts growing in fields with histories of Verticillium wilt of potato in Israel and Pennsylvania (United States), and used genotyping-by-sequencing to analyze the evolutionary history and genetic differentiation between populations of different hosts. A phylogeny inferred from 26,934 single-nucleotide polymorphisms (SNPs) in 126 V. dahliae isolates displayed a highly clonal structure correlated with vegetative compatibility groups, and isolates grouped in lineages 2A, 2B824, 4A, and 4B, with 77% of the isolates in lineage 4B. The lineages identified in this study were differentiated by host of origin; we found 2A, 2B824, and 4A only in symptomatic hosts but isolates from asymptomatic hosts (weeds, oat, and sorghum) grouped exclusively within lineage 4B, and were genetically indistinguishable from 4B isolates sampled from symptomatic hosts (potato, eggplant, and avocado). Using coalescent analysis of 158 SNPs of lineage 4B, we inferred a genealogy with clades that correlated with geographic origin. In contrast, isolates from asymptomatic and symptomatic hosts shared some of the same haplotypes and were not differentiated. We conclude that asymptomatic weeds and rotational hosts may be potential reservoirs for V. dahliae populations of lineage 4B, which are pathogenic to many cultivated hosts.

First report of Fusarium oxysporum f.sp. cubense VCG 0125 and VCG 01220 of Race 1 infecting Cavendish bananas(Musa sp. AAA) in India.

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is the most devastating disease affecting commercial and subsistence cultivation of banana (Musa spp.) worldwide. Generally, the Cavendish bananas are resistant to Foc race 1 that destroyed cv. 'Gros Michel' (AAA) and susceptible to tropical race 4 (TR4), which is causing severe epidemics in different banana-growing countries including India (Thangavelu et al. 2019). In 2019, a roving survey was conducted in major banana growing states of India such as Bihar, Uttar Pradesh, Gujarat and Tamil Nadu to assess the incidence of Fusarium wilt disease in Cavendish bananas and also to characterize the pathogens by different methods including Vegetative Compatibility Grouping (VCG) and molecular methods. The Fusarium wilt incidence in cv. Grand Naine (Cavendish group-AAA) was 6-65% in Bihar, 30-45% in Uttar Pradesh, 5-15% in Gujarat and 15- 21% in Tamil Nadu. For characterization, a total of 61 samples from the Fusarium wilt infected Cavendish bananas were collected and single spore culture of Foc was obtained. The morphological characterization revealed the presence of one to two oval- to kidney-shaped cells in false heads and sickle-shaped macroconidia and a foot-shaped basal cell. The pathogenicity was demonstrated by adopting randomized block design with five replications on cv. Grand Naine. The Koch's postulate was successfully completed by re-isolation of the inoculated Foc pathogen and characterization by PCR method. The VCG analysis carried out using nit-M testers of all known VCGs indicated the presence of VCG 0125 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Tamil Nadu (Cumbum of Theni district), VCG 01220 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Gujarat (Kamrej of Surat district,) and VCG 01213/16 from Foc samples collected from Uttar Pradesh (Siswabazar of Maharakanj district) and Bihar (Falka village of Katihar district) . The molecular confirmation of these VCGs 0125, and 01220 (Foc R1) isolates was carried out by PCR method using the primer set SIX6b_210_F and SIX6b_210_R (Carvalhais et al. 2019) for Foc R1, primer sets Foc TR4-F & Foc TR4 -R (Dita et al. 2010) for Foc TR4 and primer set Foc-1/Foc -2 (Lin et al. 2009) for Race 4. The results showed that only the primer set for Foc R1 has generated the expected amplicon size of 210 bp in the Foc isolates of VCG 0125 and 01220. Besides, the sequencing of Translation Elongation Factor (TEF) 1-α gene and BLAST searches in Genbank for the representative Foc isolates of VCG 0125 (Genbank no. MW 286800) showed 99.84% similarity to Foc R1 (KX365393.1) and Foc isolates of VCG 01220 (Genbank no. MW 286803) showed 99.69% similarity to Foc R1 (KX365413.1). Further, a phylogenetic analysis performed using the TEF1-α gene sequences showed that the Foc race 1 isolates (VCGs 0125 and 01220) from India were grouped with known Foc race 1 isolates from Tanzania and Australia. Based on the experimental results the study has confirmed the presence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India. As these VCGs are most widely distributed and do not found to infect Cavendish bananas so far (Mostert et al. 2017), this report is very important from the quarantine and management perspectives. To the best of our knowledge, this is the first report of the occurrence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India.

The potential diversity of vegetative compatibility types in the population of Cryphonectria parasitica in Turkey

AbstractThe diversity of vegetative compatibility (vc) types of chestnut blight fungus, Cryphonectria parasitica, has been reported to be very low in all of Turkey, except in the eastern Black Sea Region. However, risk for increase in vc type diversity has been pointed out regarding the emergence of new vc types as recombinants during sexual reproduction. In this study, the potential increase in the diversity of vc types in Turkish populations of C. parasitica was demonstrated by experimental mating crosses between the two dominant vc types, EU‐1 and EU‐12. Two local isolates of C. parasitica, K5‐5 (EU‐1) and A‐2490 (EU‐12), were used setting crosses on autoclaved chestnut wood. Among the offspring from the crosses, two parental (EU‐1 and EU‐12) and 14 non‐parental vc types (EU‐2, EU‐3, EU‐4, EU‐7, EU‐8, EU‐11, EU‐14, EU‐17, EU‐22, EU‐25, EU‐26, EU‐28, EU‐29 and EU‐30) were detected by a routine test based on co‐culturing. Sixteen vc genotypes were confirmed by a PCR assay using specific primers targeting the alleles of six vic loci in C. parasitica. In field studies, no evidence of new vc types as recombinants was found in western Turkey whereas 14 different vc types were detected among single ascospore isolates isolated from field‐collected perithecia, which were sampled from the eastern Black Sea region. The results of this study indicate that the potential increase in vc type diversity in Turkish population of C. parasitica is high under the conditions where C. parasitica reproduces sexually.

High phenotypic variability in the wood decay fungus Phellopilus nigrolimitatus

Genetic and phenotypic variation among individuals increases population resilience to external disturbances and enables quicker adaptation to changing environments. Since the predominant phase in the fungal lifecycle occurs belowground or within substrates, it is difficult to assess the level of variation in functional traits occurring within species in natural environments. In this study, we investigated phenotypic variation in the wood-decay fungus Phellopilus nigrolimitatus and related this to genet size, measured as vegetative compatibility groups (VCGs). Out of 321 wood samples and sporocarps collected on six logs of Picea abies , we isolated 230 dikaryotic isolates that were grouped into 53 VCGs based on culturing experiments. The number of VCGs per log varied from six to twelve, with large variation in sizes; the smallest VCG was derived from a single wood sample, while the largest was isolated across 31 wood samples stretching over 15 m. The size of the VCGs was positively correlated with the area of the spore-producing layer of the sporocarps, implying that larger genets produce more spores. Additionally, the phenotypic variation among isolates of P. nigrolimitatus was high, both in terms of growth morphology and coloration, and consistent with the delimitation of isolates into VCGs. Isolates could to a large extent be correctly assigned to the VCGs a priori based purely on culture phenotype. This study reveals that extensive phenotypic variation can be observed between fungal genets at very fine spatial scale, and calls for more studies looking at intraspecific phenotypic variation, in line with assessing genetic diversity in fungi.

Draft Genome Sequences of 20 Aspergillus flavus Isolates from Corn Kernels and Cornfield Soils in Louisiana.

Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.

Genetic Diversity of Fusarium oxysporum f. sp. cubense, the Fusarium Wilt Pathogen of Banana, in Ecuador.

The continued dispersal of Fusarium oxysporum f. sp. cubense Tropical race 4 (FocTR4), a quarantine soil-borne pathogen that kills banana, has placed this worldwide industry on alert and triggered enormous pressure on National Plant Protection (NPOs) agencies to limit new incursions. Accordingly, biosecurity plays an important role while long-term control strategies are developed. Aiming to strengthen the contingency response plan of Ecuador against FocTR4, a population biology study—including phylogenetics, mating type, vegetative compatibility group (VCG), and pathogenicity testing—was performed on isolates affecting local bananas, presumably associated with race 1 of F. oxysporum f. sp. cubense (Foc). Our results revealed that Foc populations in Ecuador comprise a single clonal lineage, associated with VCG0120. The lack of diversity observed in Foc populations is consistent with a single introduction event from which secondary outbreaks originated. The predominance of VCG0120, together with previous reports of its presence in Latin America countries, suggests this group as the main cause of the devastating Fusarium wilt epidemics that occurred in the 1950s associated to the demise of ‘Gros Michel’ bananas in the region. The isolates sampled from Ecuador caused disease in cultivars that are susceptible to races 1 and 2 under greenhouse experiments, although Fusarium wilt symptoms in the field were only found in ‘Gros Michel’. Isolates belonging to the same VCG0120 have historically caused disease on Cavendish cultivars in the subtropics. Overall, this study shows how Foc can be easily dispersed to other areas if restriction of contaminated materials is not well enforced. We highlight the need of major efforts on awareness and monitoring campaigns to analyze suspected cases and to contain potential first introduction events of FocTR4 in Ecuador.

First report of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) causing banana wilt in the Island of Mayotte.

Fusarium oxysporum f. sp. cubense (Foc) is a fungus causing Fusarium wilt of banana (Musa spp.). The fungus is divided into three races and 24 vegetative compatibility groups (VCG) of which VCG 01213/16, commonly known as Foc tropical race 4 (Foc TR4), is of particular concern. Foc TR4 severely affects Cavendish (AAA) bananas, which comprise about 50% of all bananas produced globally, as well as many varieties susceptible to the other races of Foc. The pathogen was restricted to Southeast Asia and Australia until 2012, where after it has been detected in the Middle East, Mozambique in Africa, and Colombia in South America (Viljoen et al. 2020). Here we report the first detection of Foc TR4 in the French department of Mayotte, located in the Indian Ocean. In September 2019, leaf yellowing and wilting symptoms were observed in individual plants of the banana subgroups Silk (AAB) (cv. "Kissoukari") and Bluggoe (ABB) (cv. "Baraboufaka"). The symptomatic individuals were found in private gardens in the village of Poroani in Southwest Mayotte (World Geodetic System [WGS] 12° 53' 31.83''S, 45° 8' 30.98" E). When the pseudostems of symptomatic plants were split open, dark red to brown vascular discoloration was observed. Pseudostem tissue samples were collected and identified as Foc TR4 with the real-time PCR assay developed by Aguayo et al. (2017). Sections of the pseudostem samples were surface sterilized and used to isolate the fungus on potato dextrose agar (PDA) medium. Isolates were identified as F. oxysporum based on cultural and morphological characteristics as described in Leslie and Summerell (2006), which included fluffy aerial mycelia on PDA and the presence of short monophialides conidigenous cells bearing microconidia arranged in false heads. Abundant chlamydospores were also produced on synthetic nutrient poor agar (SNA) media. Single-spored isolates were used to develop nit mutants for vegetative compatibility group (VCG) testing (Correll 1991; Puhalla 1985). The isolates were confirmed as VCG 01213/16 as formation of heterokaryons was obtained with the nit mutants of the universal Foc TR4 tester. Two VCG 01213/16 isolates were then selected for pathogenicity testing by inoculating 2-month-old tissue culture-derived Cavendish plants, using the method described by Viljoen et al. (2017). After 10 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Fusarium oxysporum was re-isolated from the inoculated plants and identified as Foc TR4/VCG 01213/16 by PCR (Dita et al. 2010; Matthews et al. 2020), thereby fulfilling Koch's postulates. Local authorities have destroyed the infected plants, and have undertaken an extensive survey to determine the distribution of Foc TR4 on the island. Three additional positive cases, identified with the real-time PCR assay of Aguayo et al. (2017), were found in the localities of Koungou ([WGS] 12° 44' 03''S, 45° 12' 08" E) and Bouéni ([WGS] 12° 54' 25''S, 45° 04' 43" E). These included infected Cavendish banana (AAA) plants (cv. "Kontriké"). This is the first time that Foc TR4 has been found on a banana variety other than Cavendish when newly detected in a country. Considering the proximity of Mayotte to other islands of the Comoros archipelago, Madagascar and the East African coast, where banana is considered an important staple, this report describes a serious threat to banana production and the livelihoods of people in the region.

Draft Genome of Fusarium oxysporum f. sp. cubense Strain Tropical Race-4 Infecting Cavendish (AAA) Group of Banana in India.

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. cubense, is the most serious pandemic disease of banana. In this study, we report the draft genome of F. oxysporum f. sp. cubense vegetative compatibility group (VCG) 01213/16 of strain tropical race 4 (TR4) that infects the Cavendish (AAA) group of banana collected from the subtropical region in India. The genome assembly of SFoc TR4 comprises 47,384,463 bp with 4,034 contigs and 15,508 protein-coding regions. Based on VCG analysis, the fungal isolate belongs to F. oxysporum f. sp. cubense TR4 but the genome sequence of SFoc TR4 shows differences in secreted-in-xylem (SIX) protein gene clusters (specifically, SIX8) in comparison with the reference genome of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense TR4.

The development of a multiplex PCR assay for the detection of Fusarium oxysporum f. sp. cubense lineage VI strains in East and Central Africa

Banana is a staple food and cash crop grown in East and Central Africa (ECA). The main banana varieties grown in ECA are the East African highland bananas (EAHB), although dessert/beer bananas such as Sukari Ndizi, Kayinja (Pisang Awak) and Gros Michel are also produced due to their high value at local markets. The Fusarium wilt fungus Fusarium oxysporum f. sp. cubense (Foc) causes disease of susceptible dessert/beer bananas, which significantly reduces yields. Banana Fusarium wilt is managed by excluding the pathogen from disease-free areas and by planting disease-resistant varieties in infested fields. Six phylogenetically closely-related vegetative compatibility groups (VCGs) of Foc, VCGs 0124, 0125, 0128, 01212, 01220 and 01222 are present in ECA, which all group together in Foc Lineage VI. Rapid and accurate detection of Foc Lineage VI strains is thus important to prevent its spread to disease-free areas. In this study, molecular markers specific to Foc Lineage VI were therefore developed. Primer sets were then combined in a multiplex PCR assay, and validated on a worldwide population of 623 known Foc isolates, other formae speciales and non-pathogenic Fusarium oxysporum isolates. The Foc Lineage VI multiplex PCR was used to identify Foc isolates collected in banana fields at five locations in Uganda and Tanzania. Foc Lineage VI DNA was detected at a concentration as low as 0.1 ng/μl, both in the absence and presence of banana DNA, and can therefore be used as an accurate diagnostic tool for Foc Lineage VI strains.