ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.     

Control of chickpea wilt (Fusarium oxysporum f.sp. ciceris) using Trichoderma spp. in Ethiopia

Thirty-two Trichoderma isolates were collected from soils grown with chickpea in central highlands of Ethiopia. The eight isolates were identified by CAB-International as Trichoderma harzianum, T. koningii and T. pseudokoningii. In in vitro tests, all Trichoderma isolates showed significant (P < 0.05) differences in their colony growth and in inhibiting the colony growth of Fusarium oxysporum f.sp. ciceris, race 3. In potted experiment, four Trichoderma isolates were tested as seed treatment on three chickpea cultivars (JG-62 susceptible, Shasho moderately susceptible and JG-74 resistant) against F. oxysporum f.sp. ciceris, race 3. The result showed that T. harzianum and unidentified Trichoderma isolate T23 significantly reduced wilt severity and delayed disease onset. The degree of wilt severity and delay of disease onset varied with chickpea cultivars. Our study revealed that biological control agents such as Trichoderma can be a useful component of integrated chickpea Fusarium wilt management.     

Biological Control Activity of Three Trichoderma Isolates Against Fusarium Wilts of Cotton and Muskmelon and Lack of Correlation with their Lytic Enzymes

The enzymatic activity and the biocontrol ability of two new isolates of Trichoderma spp. (T-68 and Gh-2) were compared in laboratory and glasshouse experiments with a previously studied T. harzianum strain (T-35). In dual culture tests with Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. vasinfectum, isolates T-68 and Gh-2 overgrew the colonies of Fusarium, whereas T-35 failed to parasitize both wilt pathogens. Under glasshouse conditions, the three isolates of Trichoderma were effective in controlling Fusarium wilt of cotton but only T-35 was effective against F. oxysporum f. sp. melonis on muskmelon. When the three Trichoderma isolates were grown on liquid media containing laminarin, colloidal chitin or F. oxysporum f. sp. melonis cell walls as sole carbon sources, maximum β-1,3-glucanase and chitinase specific activity in the culture filtrates of all fungi was reached after 72h of incubation. When culture filtrates of the three Trichoderma isolates were incubated with freeze-dried mycelium of F. oxysporum f. sp. melonis or F. oxysporum f. sp. vasinfectum, different concentrations of glucose and N-acetyl-D-glucosamine were released. Overall no correlation was found between enzymatic activity and the biocontrol capability against Fusarium wilt on muskmelon and cotton.     

Biocontrol efficacy of Trichoderma spp. against sesame wilt caused by Fusarium oxysporum f. sp. sesami

Sesame (Sesamum indicum L.) is one of the most important oilseed crops in Egypt and worldwide. It is being infected with many pathogens, among these pathogens Fusarium oxysporum f.sp. sesami (Zap.) Cast is causing severe economic losses on sesame. In this study, antagonistic capability of 24 isolates of Trichoderma spp. was assessed in vitro against F. oxysporum f.sp. sesami. Two strains; T. harzianum (T9) and T. viride (T21) were revealed to have high antagonistic effect against F. oxysporum f.sp. sesami in vitro with inhibition percentage about 70 and 67%, respectively. These two isolates proved to have high ability to control Fusarium wilt disease under greenhouse conditions. The highest reduction in disease severity was achieved with T. viride followed by T. harzianum with reduction in disease severity about 77 and 74%, respectively. This study revealed that the time of application of bioagents is a decisive factor in determining the efficacy of Trichoderma isolates to control Fusarium wilt of sesame. It was revealed that the highest reduction in the disease severity was achieved when either Trichoderma viride or T. harzianum were applied 7 days before challenging with the F. oxysporum f.sp. sesami.     

Combined application of native Trichoderma isolates possessing multiple functions for the control of Fusarium wilt disease in banana cv. Grand Naine

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is considered as a lethal disease of bananas worldwide. To manage the disease effectively, 20 rhizospheric and 43 endophytic Trichoderma isolates obtained from 12 different Foc resistant banana accessions were evaluated against Foc in vitro and in vivo. In vitro screening among Trichoderma isolates for their multiple functions (mycelial and spore germination inhibition, hydrogen cyanide, chitinolytic enzymes, non-volatile and volatile metabolites production) in suppressing Foc and promoting plant growth (IAA production and phosphate solubilisation) indicated that the multiple biocontrol actions were significantly higher in 6 isolates of rhizospheric Trichoderma and 10 isolates of endophytic Trichoderma compared to other isolates. The greenhouse evaluation of individual application of these rhizospheric and endophytic Trichoderma isolates against Fusarium wilt pathogen in cv. Grand Naine (AAA) indicated significant suppression of Fusarium wilt disease and increased plant growth characters as compared to Foc pathogen inoculated plants. However, none of these individual Trichoderma isolates recorded complete suppression of Fusarium wilt disease. Therefore, the greenhouse evaluation involving combination of rhizospheric Trichoderma sp. NRCB3 + endophytic Trichoderma asperellum Prr2 recorded 100% reduction of Fusarium wilt disease and increased plant growth parameters up to 250% when compared to individual isolates application and Foc alone-inoculated plants. Further, the field evaluation of this combination of Trichoderma isolates applied for three times: (1) at 15 days before planting, (2) second month after planting and (3) fourth month after planting resulting in significant reduction of Fusarium wilt disease and also increase in bunch weight as compared to untreated control plants. Therefore, these Trichoderma isolates may be used in combination for the effective suppression of Fusarium wilt disease in banana.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro

Trichoderma is a well-known antagonist against soilborne plant pathogens. However, the species and even various isolates have different biocontrol potential. To evaluate the antagonistic activities of Trichoderma harzianum, T. harzianum strain T100 (T100), T. viride and T. haematum against Fusarium oxysporum and F. proliferatum, we used dual culture and productions of volatile and non-volatile metabolites in three different phases in vitro. An analysis of the data in dual culture tests represented T. viride, T. haematum and T100 as effective antagonists of Fusarium while T100 was the only fungus being able to lyse the confronting mycelia. Similar results were obtained in the volatile metabolites tests also. In contrast with the two previous tests, the non-volatile metabolites produced by T. harzianum inhibited Fusarium mycelial growth the most, and T100 acted moderately. It was also clearly showed that the antagonistic effect of Trichoderma spp. was more on F. proliferatum than on F. oxysporum. Finally, because Trichoderma spp. was most effective in the second phase, we recommend to use T100 against F. proliferatum at the initial stages of infection as its mycoparasitism on F. oxysporum was observed microscopically through forming apressoria structures without any coiling around the pathogen.     

Analysis of genetic variability of Fusarium oxysporum f. sp. cepae the causal agent of basal rot on onion using RAPD markers

Genetic variability among isolates of Fusarium oxysporum f. sp. cepae was obtained from different onion-growing areas of Tamil Nadu, India. Random amplified polymorphic DNA (RAPD) analysis was carried out using 12 random primers, each of them consisting of 10 base pairs. Four out of the 12 primers were differentiated between some of the tested F. oxysporum f. sp. cepae isolates. Analysis of the genetic coefficient matrix derived from the scores of RAPD profile showed that minimum and maximum per cent similarities among the F. oxysporum f. sp. cepae isolates were in the range of 14–85%. Cluster analysis, using the unweighted pair-group method with arithmetic average, clearly separated the isolates into two clusters (A and B) confirming the genetic diversity among the isolates of F. oxysporum f. sp. cepae from onion.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Characterization of Fusarium oxysporum f.sp. cepae from Onion in Turkey Based on Vegetative Compatibility and rDNA RFLP Analysis

Seventy‐five isolates of Fusarium oxysporum f.sp. cepae, the causal agent of basal plate rot on onion, were obtained from seven provinces of Turkey. The isolates were characterized by vegetative compatibility grouping (VCGs) and restriction fragment length polymorphism (RFLP) analysis of the nuclear ribosomal DNA intergenic spacer region (IGS). Forty‐eight vegetative compatibility groups were found, each containing a single isolate. Only one isolate formed strong heterokaryons with the reference isolates of VCG 0423. Five isolates were heterokaryon self‐incompatible. Restriction fragment analysis with six different enzymes revealed 13 IGS types among 75 F. oxysporum isolates from Turkey as well as 16 reference isolates from Colorado, USA. The majority of single‐member VCGs produced identical RFLP banding patterns with minor deviations, considerably different from those of the reference VCG isolates. These results suggested that isolates of F. oxysporum f.sp. cepae in Turkey derived from distinct clonal lineages and mutations at one or more vegetative compatibility loci restrict heterokaryon formation.     

Presence of Fusarium oxysporum f. sp. melonis Race 1 in Soils Cultivated with Melon in the State of Colima (Mexico)

During years 2001, 2002 and 2003 the gravity of the Fusarium wilt in 1000 hectares of melon culture was evaluated in Colima (Mexico). In spite of the soil disinfections with methyl bromide, the losses could reach 25% of the final production. The analysis of 4 soil samples from the fields with ill plants, in a selective medium for Fusarium, allowed to detect the presence of F. oxysporum. By means of the presented technique “soil phytopathometry”, 31 isolates of F. oxysporum f. sp. melonis were obtained from the soil samples. The isolates were inoculated on melon plants to evaluate their pathogenicity. The 31 isolates inoculated, produced the symptoms of chlorosis and wilting, in melon cultivars that allowed us to affirm that all isolates were race 1 of F. oxysporum f. sp. melonis. Being this the first news of the presence of F. oxysporum f. sp. melonis in the state of Colima (Mexico).     

RAPD based genetic diversity among different isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp<Emphasis Type="Italic">. lycopersici</Emphasis> and their comparative biocontrol

Fusarium wilt of tomato (Solanum lycopersicum Mill.) caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) W. C. Snyder and H. N. Hans (Fol.), is most serious and versatile pathogen. Chemical control of disease is not satisfactory and biological control is an attractive and potential alternative to the use of chemicals to control fusarium wilt of tomato. No any bioagent is universally effective everywhere therefore, search for potential biocontrol agent is continuous process and mandatory for several and individual ecological niches. In this experiment biocontrol efficacy of five species of Aspergillus and five species of Trichoderma were evaluated in vitro against Fusarium oxysporum f. sp. lycopersici. In both the experiments (dual culture and culture filtrates) T. harzianum was found to be highly effective against the isolates of Fol. followed by A. niger biocontrol potential of A. terreus is least among all the isolates tested. Culture filtrates obtained from A. luchuensis exerted least inhibition of Fol. The most sensitive isolate of Fol. against all the antagonists tested was identified as IIVR-2 (Fol. 9). Inherent diversity among Fol. isolates, from different tomato growing regions in India, was determined using RAPD primers. The genetic similarity coefficients ranged from 0.20 to 0.96, indicating that no any two or more isolates were 100% similar. RAPD profiles revealed up to 20% genetic diversity among ten isolates of Fusarium oxysporum f. sp. lycopersici.     

Potential of microsatellites to distinguish four races of Fusarium oxysporum f. sp. ciceri prevalent in India

Fusarium oxysporum f. sp. ciceri, the causal agent of chickpea wilt, is an important fungal pathogen in India. Thirteen oligonucleotide probes complementary to microsatellite loci, in combination with 11 restriction enzymes, were used to assess the potential of such markers to study genetic variability in four Indian races of the pathogen. Hybridisation patterns, which were dependent upon both the restriction enzyme and oligonucleotide probe used, revealed the presence of different repeat motifs in the F. oxysporum f. sp. ciceri genome. Among the restriction enzymes used, hexa-cutting enzymes were more informative than tetra- and penta-cutting enzymes, whereas tetranucleotide and trinucleotide repeats yielded better hybridisation patterns than dinucleotide repeats. Dependent upon the levels of polymorphism detected, we have identified (AGT)₅, (ATC)₅ and (GATA)₄ as the best fingerprinting probes for the F. oxysporum f. sp. ciceri races. The distribution of microsatellite repeats in the genome revealed races 1 and 4 to be closely related at a similarity index value of 76.6%, as compared to race 2 at a similarity value of 67.3%; race 3 was very distinct at a similarity value of 26.7%. Our study demonstrates the potential of oligonucleotide probes for fingerprinting and studying variability in the F. oxysporum f. sp. ciceri races and represents a step towards the identification of potential race diagnostic markers. Received: 12 March 2000 / Accepted: 14 April 2000     

Fusarium oxysporum Forms Heterokaryons with Fusarium redolens

Vegetative compatibility among three isolates of Fusarium oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens from diseased lupins was investigated. Pairings between five mutants originated from each isolate revealed two compatibility groups. The first VCG comprised race 1 of F. oxysporum f. sp. lupini and one isolate of F. oxysporum var. redolens; the second VCG comprised race 2 of F. oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens. Heterokaryon formation was observed in many pairings involving mutants of both taxa. These findings provide evidence of the conspecificity of these two taxa and they support Gordon 's classification (1952) according to which F. redolens is actually F. oxysporum.     

Molecular phylogeny of Fusarium oxysporum species complex isolated from agricultural soils in Iran

Abstract

Members of Fusarium oxysporum species complex (FOSC) are economically most important plant pathogenic fungi. Until now, the classification of FOSC members in Iran is not well described. So, the objective of the current research was to study the phylogenetic diversity of FOSC strains recovered from agricultural soils in Iran. A total of 45 isolates belonging to the FOSC were recovered from agricultural soils in Iran. The identification of the members of F. oxysporum f. sp. vasinfectum (Fov) and F. oxysporum f. sp. ciceris (Foc) was confirmed molecularly using Fov-eg-f/Fov-eg-r and Foc0-12f/Foc0-12r primers, respectively. F. redolens isolates were distinguished from other FOSC using Redolens-F/Redolens-R primers. Comparisons of DNA sequence data from a portion of the tef1 gene revealed all isolates belonged to Fov, Foc, F. commune and F. redolens. This is the first in-depth report on molecular identification of FOSC and related species isolated from agricultural soils in Iran.     

Fusarium Species Isolated from Common Weeds in Eggplant Fields and Symptomless Hosts of Fusarium oxysporum f. sp. melongenae in Turkey

Thirteen species of weed plants were collected between May and September in 2010 and 2011 from eggplant fields representing 11 distinct locations covering a wide geographical area of Turkey. Weeds are potential hosts of many plant pathogens and may not exhibit disease symptoms when colonized. Fusarium spp. were isolated from five monocotyledonous species and eight dicotyledonous species. A total of 212 isolates recovered from weeds were assigned to eight Fusarium species on the basis of morphological characteristics. F. oxysporum was the most frequently isolated species (29.7%), followed by F. solani (19.8%), F. graminearum (13.7%), F. verticillioides (12.7%), F.equiseti (9.9%), F. avenacearum (8.0%), F. proliferatum (3.8%) and F. subglutinans (2.4%). The F. oxysporum isolates from different weed hosts were characterized by means of pathogenicity and vegetative compatibility grouping (VCG) tests. Among these, 29 isolates were found to be pathogenic to eggplant cv. Kemer and re‐isolated as Fusarium oxysporum Schlecht. f. sp. melongenae (Fomg) as evidenced. These isolates from weed hosts were assigned to VCG 0320. This study is the first report of Fomg isolated from weeds in eggplant fields in Turkey. None of the weed species tested showed symptoms of wilting in pot experiments, and F. oxysporum was isolated with greater frequency from all inoculated weeds. The results of this study indicate that several weed plants may serve as alternative sources of inoculum for Fomg, during the growing season.     

Microsatellite marker-based detection of Fusarium wilt pathogens of Psidium guajava L.

Wilt of Psidium guajava L., incited by Fusarium oxysporum f. sp. psidii and Fusarium solani is a serious soil-borne disease of guava in India. Forty-two isolates each of F. oxysporum f. sp. psidii (Fop) and F. solani (Fs) collected from different agro climatic zones of India showing pathogenicity were subjected to estimate the genetic and molecular characterisation in terms of analysis of microsatellite marker studies. Out of eight microsatellite markers, only four microsatellite markers, viz. MB 13, MB 17, RE 102 and AY212027 were amplified with single band pattern showing the character of identical marker for molecular characterisation and genetic identification. Microsatellite marker MB 13 was amplified in F. oxysporum f. sp. psidii and F. solani isolates. Product size of 296 bps and 1018 bps were exactly amplified with a single banding pattern in all the isolates of F. oxysporum f. sp. psidii and F. solani, respectively. Microsatellite markers, viz. MB 17, RE 102 and AY212027 were also exactly amplified with a single banding pattern. MB 17 was amplified in F. oxysporum f. sp. psidii isolates with a product size of 300 bp. RE 102 and AY212027 were amplified in F. solani isolates with the product size of 153 bp and 300 bp, respectively. Therefore, amplified microsatellite marker may be used as identifying DNA marker.     

Molecular Characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD Markers on Eggplant

Fusarium oxysporum f. melongenae is a major soil-borne pathogen of eggplant (Solanum melongena). ISSR and RAPD markers were used to characterize Fusarium oxysporum f. melongenae isolates collected from eggplant fields in southern Turkey. Those isolates were not pathogenic to tomato. Pathogens were identified by their morphology, and their identity was confirmed by PCR amplification using the specific primer PF02-3. The isolates were classified into groups on the basis of ISSR and RAPD fingerprints, which showed a level of genetic specificity and diversity not previously identified in Fusarium oxysporum f. melongenae, suggesting that genetic differences are related to the pathogen in the Mediterranean region. The primers selected to characterize Fusarium oxysporum f. melongenae may be used to determine genetic differences and pathogen virulence. This study is the first to characterize eggplant F. oxysporum species using ISSR and RAPD.     

Molecular diversity of Fusarium oxysporum causing rot diseases of vanilla in south India

Incidence of root, stem and beans rot of vanilla (Vanilla planifolia Andrews) caused by Fusarium oxysporum Schlecht was surveyed in vanilla growing areas of south India during December 2008. The incidence of the disease varied from 1 to 100% in different locations. A total of 60 isolates of F. oxysporum were obtained from diseased samples, and nine morphologically different isolates were taken for molecular characterization using Randomly Amplified Polymorphic DNA (RAPD) markers to study the genetic variability if any, among them. PCR amplification of total genomic DNA with random oligonucleotide primers generated unique banding patterns depending upon primers and isolates. Nine oligonucleotide primers were selected for the RAPD assays, which resulted in 384 bands for nine isolates of F. oxysporum. The number of bands obtained was entered into a NTSYS and the results showed that the variability among the pathogen isolates was moderate. The nine isolates studied were grouped into single major cluster at 0.66 similarity index. Hence, it is inferred that F. oxysporum infecting vanilla in south India consists of a single clonal lineage with a moderate level of genetic diversification.