New Hybrid Strains via Intraspecific Protoplast Fusion of the Entomopathogenic Fungi Lecanicillium spp.

The entomopathogenic fungal genus Lecanicillium Gams and Zare (formerly classified as the species Verticillium lecanii) includes species that are highly pathogenic to many insect genera. In this study, we identified six Lecanicilliumspp. isolated strains (designated as V1-V6) belonging to L. lecanii (V1, V3 and V5) and L. attenuatum (V2, V4 and V6). In addition, these strains were used to obtain new strains via protoplast fusion, and nit mutants were used for protoplast selection. Genetic recombination of the hybrid strains was determined using the random amplified polymorphic DNA(RAPD) technique. We obtained nine stable fusant strains from 176 new hybrid strains, which were termed V12-10, V14-3, V16-4, V23-6, V25-8, V34-14, V36-5, V45-16 and V56-7. Morphological characteristics varied between the hybrid and parental strains. Genomic DNA analysis of the fusants also showed genetic recombination. The median lethal concentration (LC50) for the fusants were lower than that for parental strains, and the median survival time (LT50) for the fusants were reduced compared with that for parental strains. Thus, these results showed that we produced new, more virulent hybrid Lecanicillium spp. strainsas biological control agents via intraspecific protoplast fusion.

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.

The Population of Fusarium oxysporum f. sp. lactucae in California and Arizona

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae, is now found in all major lettuce producing regions in California and Arizona. The population structure of F. oxysporum f. sp. lactucae in California and Arizona was characterized based on somatic compatibility and sequences of the translation elongation factor 1-α gene (EF-1α) and rDNA intergenic spacer region (IGS). In this study, 170 isolates were tested for somatic compatibility based on heterokaryon formation, using complementary nitrate nonutilizing (nit) mutants. Five subgroups (A to E) of somatic compatibility group 0300 were identified. Isolates associated with the same subgroup had a strong complementation reaction, whereas reactions between isolates of different subgroups were weak or delayed. An isolate from the first known infestation of Fusarium wilt of lettuce in California was associated with subgroup A, which predominated among isolates in our collection. Isolates representative of each subgroup were confirmed to be associated with race 1, based on the reaction of differential lettuce cultivars. It is possible that somatic compatibility subgroups B to E of F. oxysporum f. sp. lactucae were derived from subgroup A, as a consequence of somatic mutations affecting compatibility. If so, subgroups of F. oxysporum f. sp. lactucae may represent an intermediate step in divergence that will lead to clearly separable compatibility groups. Sequences of EF-1α and IGS were both identical for 58 isolates of F. oxysporum f. sp. lactucae that represented all somatic compatibility subgroups and locations from which isolates were obtained, indicating that subgroups were derived from the same clonal lineage (VCG 0300).

Kompatibilitas Vegetatif Fusarium oxysporum dari Beberapa Tanaman Inang

ABSTRACTVegetatif compatibility of Fusarium oxysporum on various hostsMany strains or race of Fusarium oxysporum can be grouped based on compatibility reproduction from a variety of different strains called Vegetative Compatibility Group (VCG). This study was aimed to determine how the grouping of several isolates of F. oxysporum and grouping of several hosts of the fungus by vegetative compatibility group. Fusarium oxysporum isolated from chickpea plants that showed symptoms of fusarium wilt. The isolates of F. oxysporum of chili and tomatoes obtained from the culture collections of Mycology Laboratory of IPB. Stages of vegetative compatibility testing assayed through recovery of nit mutants, the identification of phenotype of nit mutant, and complementation test. There are 29 mutants isolated from the isolates of F. oxysporum. Nit1 mutant was obtained from all isolates of beans, tomatoes and peppers. NitM and Nit3 mutant isolates were obtained from chickpea 4 and chili sequentially. Two VCG and one single self compatibility (SSC) were assayed from isolates of F. oxysporum based on complementation testing.Keywords: Beans, Fusarium wilt, Nit mutant, SSC, VCGABSTRAKJamur Fusarium oxysporum memiliki banyak forma spesialis dan ras. Jamur ini dapat dikelompokkan berdasarkan kompatibilitas reproduksi dari berbagai strain yang berbeda disebut dengan vegetative compatibility group (VCG). Penelitian ini bertujuan untuk mengetahui cara pengelompokkan F. oxysporum dan pengelompokkan jamur tersebut dari beberapa inang berdasarkan kelompok kompatibilitas vegetatifnya. Isolasi F. oxysporum dilakukan dari tanaman kacang panjang yang menunjukkan gejala layu fusarium. Isolat F. oxysporum dari cabai dan tomat berasal dari koleksi Laboratorium Mikologi IPB. Tahapan pengujian kompatibilitas vegetatif melalui pembiakan nit mutan, identifikasi fenotipe nit mutan, dan pengujian komplementasi. Isolasi mutan F. oxysporum didapatkan 29 mutan. Mutan nit1 didapatkan dari semua isolat yang diperoleh dari semua inang yang berbeda yaitu kacang panjang, tomat dan cabai. Mutan nitM hanya didapatkan dari isolat kacang panjang 4 dan mutan nit3 hanya didapatkan dari isolat cabai. Berdasarkan uji komplementasi F. oxysporum yang diuji terdiri dari dua VCG dan satu single self compatibility (SSC).Kata Kunci: Kacang panjang, Layu fusarium, Nit mutant, SSC, VCG

An efficient method for selecting stable tester strains of vegetative compatibility groups in Verticillium dahliae

The way compatible pairs of nitrate non-utilizing mutants (nit) are selected is usually not explained and remains unclear whether these pairs are representative for an isolate or strain. In addition, tester strains of Verticillium dahliae vegetative compatibility groups (VCGs) cross-react with at least one pair of tester strains of another VCG, and although it is a common knowledge of scientists working with the fungus that reversion of nit mutants to wild type occurs far too often, this fact is rarely mentioned in papers. To overcome the above problems, a protocol was developed for the generation of large number of nit mutants from any given isolate and to ensure that compatible pairs of mutants are indeed stable and the most frequent within the putative tester mutants produced from each isolate. Thus, we provide a reproducible and objective way of selecting V. dahliae tester strains for each isolate and VCG. Although VC grouping is based on the formation of stable heterokaryons, we demonstrate in this work that cross-reactions cannot be eliminated and that strict genetic barriers between two main VCG groups are absent in V. dahliae.

Identification of Aspergillus flavus Isolates as Potential Biocontrol Agents of Aflatoxin Contamination in Crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops.

Fusarium verticillioides from sugarcane, vegetative compatibility groups and pathogenicity

Sugarcane plant samples affected by the disease pokkah boeng were collected from the major sugarcane producing areas in Iran. Fifty isolates of Fusarium verticillioides were recovered from diseased sugarcanes. Results of pathogenicity tests revealed that all isolates were pathogenic to sugarcane. Four hundred and eighty-five nitrate non-utilising mutants were generated from F. verticillioides isolates. Media used for nit mutant generation included potato dextrose agar, minimal medium and Czapeck agar amended with 1.8%, 2%, 2.5%, and 3% potassium chlorate. Nit mutants were divided into three phenotypic (nit1, nit3, and nitM) classes based on their growth on different nitrogen sources in the culture media. Among the isolates tested, 280, 115 and 90 nit1, nit3, and nit M were generated, respectively. Nit mutants were used to force the heterokaryon formation to determine the distribution of VCGs and their relation to pathogenicity and geographic origin. Forty-eight VCGs of F. verticillioides were isolated from sugarcane. Forty-six of the VCGs were represented by a single isolate, whereas the remaining two comprised two isolates. None of the VCGs was common.

Vegetative Compatibility Groups and Parasexual Segregation in Colletotrichum acutatum Isolates Infecting Different Hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.