Biochemical and molecular characterization of wild-type and fused protoplasts of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Protoplasts were isolated from two isolates each of Beauveria bassiana and Metarhizium anisopliae using lysing enzymes. Intra- and intergeneric protoplast fusion has been carried out using 40% polyethylene glycol. The fused protoplasts of B. bassiana and M. anisopliae have been regenerated on Czapek–Dox agar media, and a total of four fusants were selected for further studies. An increase in proteinase and chitinase enzyme activity was recorded with all fusants as compared to the wild-type isolates. To understand the nature of recombination process, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were carried out on genomic DNA of fused and wild-type isolates. The present study demonstrates the scope and significance of the protoplast fusion technique as a rapid consistent method for identification of B. bassiana and M. anisopliae fused and wild-type isolates based on the banding pattern of RAPD and RFLP that can be reliably used ahead for further applications on these species.     

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morpho‐pathological and Molecular Characterization of Bipolaris oryzae in Rice (Oryzae sativa)

Fifty isolates of Bipolaris oryzae from rice were characterized morpho‐pathologically and molecularly. Based on colony morphology and growth pattern on PDA, these isolates were grouped into four categories: black with suppressed growth (21 isolates), black with cottony growth (16 isolates), black with fluffy growth (12 isolates) and white with cottony growth (1 isolate). The frequency of the black and suppressed type was the highest (42%) with maximum aggressiveness (mean spore count of 1854/cm²), whereas the white and cottony growth isolate had lowest frequency (2%) and aggressiveness (548/cm²). Thirteen B. oryzae isolates (four isolates from Groups I, II and III and one isolate from Group IV) were further tested for their variability with random amplified polymorphic DNA (RAPD) primers. Twenty RAPD primers were screened, of which 10 gave amplification; however, only six primers gave reproducible results. Based on the molecular similarity of the RAPD profiles, the isolates were grouped in to three major clusters and maximum linkage distance between them was determined as 0.29 units. This study establishes the variability among B. oryzae isolates.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Discovery of a North American genetic variant of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">anisopliae</Emphasis> pathogenic to grasshoppers

A genetic variant of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, isolated from a soil in Alberta, Canada, from a location with a history of severe grasshopper infestations, was evaluated for pathogenicity in bioassays of living grasshoppers. Mortality in treated individuals drawn from a laboratory colony was 99% (LT₅₀ = 6.7 days, LT₉₀ = 9.6 days) at 12 days post-inoculation compared to 100% (LT₅₀ = 4.1 days, LT₉₀ = 5.8 days) mortality at 8 days in insects exposed to a commercial isolate of M. anisopliae var. acridum (IMI 330189). Experimental infection of field-collected grasshoppers under laboratory conditions with the native isolate of M. anisopliae var. anisopliae resulted in 100% (LT₅₀ = 4.4 days, LT₉₀ = 5.4 days) mortality attained within 7 days compared to 100% (LT₅₀ = 4.7 days, LT₉₀ = 6.3 days) mortality in 9 days in insects treated with M. anisopliae var. acridum. Amplification of fungal genomic DNA from the indigenous isolate with primers for the specific detection of M. anisopliae var. anisopliae produced a product almost 300 bp larger than expected based on previously known isolates. This is the first demonstration of a highly virulent, indigenous non-chemical control agent of grasshoppers in North America. GenBank Accession Nos. DQ342236, DQ342237.     

Characterization of Mycoviruses and Analyses of Chitinase Secretion in the Biocontrol Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae. Received: 25 September 2001 / Accepted: 1 February 2002     

Clonal Variability and Its Relevance in Generation of New Pathotypes in the Spot Blotch Pathogen, <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

Spot blotch pathogen Bipolaris sorokiniana of wheat was investigated with threefold objectives: to establish a relationship between morphological and pathological variability of isolates, identify clonal genotype(s) acting as a source for the generation of new variability, and to determine the mechanism of generation of such variability in the pathogen. Isolates were collected from the leaves and seeds of field-grown wheat crop at four different sites in eastern Gangetic plains of India. Eighty-six clonal isolates derived from a single isolate (gray with white patches, Group III), which segregated in an equal proportion of parental and nonparental types, were studied. Morphological characters—i.e., colony morphology, growth rate, and sporulation—were studied along with disease-causing ability of the isolate clones. Clonal isolates were grouped into three categories. Microscopic analysis of nuclei was done to determine the causes of such variability. Morphological variability appeared to be related to the pathological variability. The isolate having epidemic potential appeared different than that acting as the reservoir for variability. The cause of such variability could be attributed either to hyphal fusion and heterokaryosis, nuclear migration and occurrence of multinucleate state, or a combination of these factors. Random Amplified Polymorphic DNA (RAPD) assay suggested that the unique fragments for different groups could be utilized as molecular markers to identify the isolates of specific groups.     

Activity of glutathione S‐transferase and esterase enzymes in Helicoverpa armigera (Hübner) after exposure to entomopathogenic fungi

Helicoverpa armigera, a polyphagous insect of crops and vegetables, is acquiring resistance against many commercial insecticides. The present study shows variations in the activity of two detoxification enzymes, namely esterase and glutathione S‐transferase (GST), in H. armigera after exposure to different isolates of entomopathogenic fungi. After treatment of larvae with the different isolates (Day 0), samples were collected on three days (Days 3, 5 and 7) for enzyme analysis. High GST activity was found in samples of hemolymph, intestine and fat bodies of H. armigera following treatment with Beauveria bassiana (isolate Bb‐08), Metarhizium anisopliae (isolates Ma‐11.1 and Ma‐4.1), and Isaria fumosorosea (isolates If‐02 and If‐2.3). High esterase activity was recorded in samples of the intestine and fat bodies on various days after treatment, whereas increased esterase activity in hemolymph was noted only in samples from Day 5 after treatment with M. anisopliae (Ma‐4.1). The detection of high GST and esterase activity demonstrates the possibility of the development of resistance against these microbial control agents in H. armigera.     

Chitinolytic enzymes from the newly isolated <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg⁻¹, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH₂PO₄ and K₂HPO₄); NaCl, 0.25%; MgSO₄, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD₅₀ 0.60 U ml⁻¹ at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.     

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) rhizosphere

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099^T), Pseudomonas aureofaciens (NCIM2026^T), and Pseudomonas aeruginosa (MTCC2582^T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH₄)₂SO₄ precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn²⁺, Cu²⁺, and Ni²⁺ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.     

Effects of carbon and nitrogen sources on the induction and repression of chitinase enzyme from<Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> isolates

Metarhizium anisopliae, an entomopathogenic hyphomycete, is being used effectively in Integrated Pest Management (IPM) system. Foliar application of these fungi is quite satisfactory as it invades its host by adhering to insect cuticles and formation of penetration structures called appresoria, which produces various extracellular enzymes, including chitinase that causes the insect cuticle breaching. The induction and repression mechanism of chitinase activity is not entirely understood and activity of this enzyme is different in response to different carbon and nitrogen sources. This report illustrates the effect of two carbon sources viz. colloidal chitin and dextrose and a nitrogen source, yeast extract on the chitinase production of fourteenM. Anisopliae isolates. The chitinase activity varied among the isolates and the different media used. A high enzymatic activity was observed in the medium containing an extra nitrogen source (yeast extract) followed by the medium containing colloidal chitin as a sole source of carbon and nitrogen. The exochitinase activity and the chitinase activity gel were also studied for the isolates showing high chitinase enzyme production. An array of chitinase isozymes were observed on chitinase activity gel with a common 14.3 kDa enzyme for all the isolates.     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

Influence of various nitrogen and carbon sources on the production of pectolytic,cellulolytic and proteolytic enzymes byAspergillus niger

Three isolates ofAspergillus niger produced polygalacturonase (PG) and pectin methyl galacturonase (PMG) in the presence of organic and inorganic nitrogen sources. Complete inhibition of PG PMG cellulase (C_x) and proteinase synthesis was found in the presence of cystine in all isolates. Maximum biomass was found in sodium nitrate whereas no isolate could grow in the presence of cystine. A correlation between biomass and enzyme production could be obtained when sodium nitrate and cystine were added to the medium separately. All isolates produced pectic cellulolytic and proteolytic enzymes in the presence of various native carbon sources. Sodium polypectate was found to be the best carbon source for the production of PG and PMG; pectin inhibited completely the production of PG and PMG. Maximum cellulase production was brought about by cotton in all three isolates. Maximum proteinase production was observed with gelatin which served as poor substrate for fungal growth. Sucrose supported maximum fungal growth in comparison with all other native carbon sources. The increased production of pectolytic cellulolytic and proteolytic enzymes in the presence of sodium polypectate reflected a stimulation rather than an induction of synthesis of these enzymes.     

Formation of interspecies fusants of <Emphasis Type="Italic">Agaricus bisporus</Emphasis> and <Emphasis Type="Italic">Agaricus bitorquis</Emphasis> mushroom by protoplast fusion

Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons). These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons. Antifungal markers were developed for selection of fusants.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.