Development of PCR-Based Race-Specific Markers for Differentiation of Indian Fusarium oxysporum f. sp. cubense, the Causal Agent of Fusarium Wilt in Banana

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL−1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.

Resistance of Three Superior New Banana Varieties on Several Vegetative Compatibility Groups Fusarium oxysporum f. sp. cubense

Balai Penelitian Tanaman Buah (Balitbu) Tropika telah melepas tiga varietas unggul baru (VUB) pisang dengan keunggulan masing-masing. Dalam menunjang keberhasilan pengembangan varietas unggul baru tersebut perlu diketahui informasi tentang ketahanannya terhadap beberapa Vegetative Compatibility Groups (VCGs) Fusarium oxysporum f. sp. cubense (Foc). Tujuan penelitian ini ialah menentukan ketahanan tiga VUB pisang terhadap beberapa VCGs Foc di rumah kasa. Penelitian dilakukan di laboratorium proteksi tumbuhan dan rumah kasa Balitbu Tropika di Solok, dari bulan September 2016 sampai bulan Desember 2017. Rancangan yang digunakan ialah acak kelompok 21 perlakuan dan 3 ulangan, masing-masing perlakuan terdiri atas 10 tanaman. Sebanyak 3 VUB pisang (Ketan 01, Kepok Tanjung dan Raja Kinalun) masing-masing diinokulasi dengan 7 VCGs Foc: yaitu VCG 0120/15, 0123, 0124/5, 0126, 01218, 01213/16 dan 01219. Hasil penelitian menunjukkan bahwa tiga VUB pisang menunjukkan respons ketahanan yang bervariasi terhadap isolat Foc dari berbagai VCGs dalam ras 1 dan ras 4. Ketahanan dipengaruhi oleh kompatibilitas masing-masing VCGs dari isolat Foc dengan varietas pisang. Varietas Ketan 01 (AA) mempunyai respons agak tahan dan tahan terhadap semua VCGs Foc yang diuji kecuali dengan VCG 0124/5 (ras 1) dan VCG 01213/16 (ras 4 tropis). Kepok Tanjung (ABB/BBB) rentan dan sangat rentan terhadap semua VCGs Foc, Raja Kinalun (ABB) memberikan respons tahan dan sangat tahan terhadap semua isolat VCGs Foc kecuali dengan isolat Foc VCG 0124/5 (ras 1) responsnya sangat rentan. Hasil penelitian ini bermanfaat sebagai informasi awal pengembangan varietas unggul baru pisang di Indonesia.

Current Status of Fusarium and Their Management Strategies

Fusarium spp. is one of the most economically important plant pathogens causing a wide range of plant diseases with significant crop losses globally. Fusarium wilt is a major problem all over the world. Fusarium oxysporum, Fusarium solani, Fusarium fujikuroi are economic importance species in worldwide. Fusarium solani causing disease in many agriculturally crops and favored by high temperatures and warm moist soils. The fungus produces three types of asexual spores; microconidia, macroconidia and chlamydospores serve as propagules in infecting host plants and found endophytes and saprophytes. The color of the colony, length and shape of the macroconidia, the number shape of microconidia and the presence or absence of chlamydospores are key features for the differentiation of Fusarium species. Pathogens, forms over 100 formae speciales cause disease in dicot and monocot plant species and infecting a variety of hosts. Vegetative compatibility Groups (VCG) is used to differentiate their races. Resistant cultivars and bio-control agents (Trichoderma spp., and Psedomonas spp.) have been used to manage the disease.

Molecular characterization and vegetative compatibility groups of Fusarium spp. pathogenic to Ilex paraguariensis A. St.-Hil.

Nas regiões Centro-Oeste e Sul do Brasil, a cultura da erva-mate (Ilex paraguariensis) constitui-se uma importante fonte de extrativismo florestal, além de diversificar a renda, principalmente na agricultura familiar. A erva-mate, como outras espécies florestais, é infectada por diversos patógenos, como o gênero Fusarium, causador da podridão-de-raízes. Os patógenos possuem ampla variabilidade genética, mesmo dentro de um único gênero e dentro de uma única espécie, resultado de adaptações ao meio em que vivem. Este estudo teve como objetivo avaliar o sequenciamento das regiões do genoma ITS e β-tubulina na identificação em nível de espécie dos isolados de Fusarium spp. patogênicos à erva-mate, e identificar a formação de grupos de compatibilidade vegetativa (VCG) entre os isolados de Fusarium spp. patogênicos à erva-mate. O sequenciamento das regiões ITS e β-tubulina consolidou a separação entre os sete isolados patogênicos em F. solani e F. oxysporum. Para esses isolados, a técnica para obtenção de mutantes nit foi adequada para a classificação fenotípica, fundamentais nos testes de VCG para espécies de Fusarium, sendo obtidos cinco VCG.

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.

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.

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.

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

ABSTRACT 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.

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 U.S. A multi-locus phylogenetic analysis with three genes was conducted with a collection of isolates from celery (23) and non-celery (29) hosts to evaluate their taxonomic position within C. acutatum sensu lato. The three DNA regions used for phylogenetic analysis included the introns of glutamine synthase (GS) and glyceraldehyde-3-phosphate dehydrogenase (GPDH), and the partial sequence of the histone3 (his3) gene. Moreover, celery and non-celery isolates were evaluated for vegetative compatibility and pathogenicity on celery. Culture filtrates from celery and non-celery 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 the population was not clonal. Twenty two of 23 isolates of C. fioriniae from celery 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 (IAA).

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.