Characterization of Rhizoctonia solani associated with soybean in Brazil

Rhizoctonia solani causes pre- and post-emergence damping-off, root and hypocotyl rot and foliar blight in soybean. Foliar blight has resulted in yield losses of 31-60% in north and northeast Brazil. The aim of this study was to characterize isolates of R. solani associated with soybean in Brazil. Among 73 Rhizoctonia isolates examined, six were binucleate and 67 were multinucleate. The multinucleate isolates were characterized according to hyphal anastomosis reaction, mycelial growth rate, thiamine requirement, sclerotia production, and RAPD molecular markers. Four isolates that caused hypocotyl rot belonged to AG-4 and using RAPD analysis they grouped together with the HGI subgroup. Another isolate that caused root and hypocotyl rots was thiamine auxotrophic, grew at 35 °C, and belonged to AG-2-2 IIIB. All 62 isolates that caused foliar blight belonged to AG-1 IA. RAPD analysis of R. solani AG-1 IA soybean isolates showed high genetic similarity to a tester strain of AG-1 IA, confirming their classification. The teleomorph of R. solani, Thanatephorus cucumeris was produced in vitro by one AG-1 IA isolate from soybean. The AG-4 and AG-2-2 IIIB isolates caused damping-off and root and hypocotyl rots of soybean seedlings cv. 'FT-Cristalina', under greenhouse conditions. The AG-2-2 IIIB isolate caused large lesions on the cortex tissue, that was distinct from the symptoms caused by AG-4 isolates. The AG-1 IA isolates caused foliar blight in adult soybean plants cv. 'Xingu' under the greenhouse and also in a detached-leaf assay.

Host specificity of a Brazilian isolate of Alternaria cassiae (Cenargen CG593) under greenhouse conditions

Several Alternaria cassiae isolates were recovered from diseased sicklepod plants (Senna obtusifolia) in the southern regions of Brazil. A representative isolate (Cenargen CG593) was tested for its host range under greenhouse conditions. The fungus promoted symptoms in sicklepod, cassava (Manihot dulce), tomato (Lycopersicon esculentum) and eggplant (Solanum melongena) when tested at a spore concentration of 10(6) spores ml(-1). When the plants were inoculated with a suspension of 10(5) spores ml(-1) and held at a dew period of 12 h (cassava) or 18 h (tomato and eggplant), the plants showed symptoms of the disease, but they recovered and continued their normal vegetative growth. These results show that the fungus A. cassiae is safe to use for the control of S. obtusifolia under Brazilian conditions, because it did not cause excessive damage in the three plants tested.

Production technology for entomopathogenic fungus using a biphasic culture system

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of Metarhizium anisopliae fungus on off-host Rhipicephalus (Boophilus) microplus from tick-infested pasture under cattle grazing in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

Background: the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).Results: Populations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Phi(ST) = 0.257, significant at P < 0.05) but not for locus pP42F (Phi(ST) = 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3.Conclusion: the two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment 1), we analyzed two antigen lots of two human isolates (Bt1 and Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh and Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment 2), we compared the antigenic profile of three antigen hatches from three human isolates (Bt1, Bt2 and Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment 1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment 2, the reference system for P brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 106, 107 and 108 conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36-65.58% and increased the mortality rate of inoculated larvae by 0.8-70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69-47.80%, egg mass weight by 4.71-53.87%, estimated reproduction by 8.3-60.62%, egg production index by 5.03-54.20%, percent larval hatching by 0.27-13.96%, and the mortality rate of treated females was increased by 96.60-100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.

Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae)

An isolate of Curvularia sp. was obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado. The ethyl acetate extract from culture of this fungus afforded two benzopyran derivatives: (2′S)-2-(propan-2′-ol)-5-hydroxy-benzopyran-4-one (2) and 2,3-dihydro-2-methyl-benzopyran-4,5-diol (4); and two known benzopyrans: 2-methyl-5-methoxy-benzopyran-4-one (1) and (2R)-2,3-dihydro-2-methyl-5-methoxy- benzopyran-4-one (3). The structures of 2 and 4 were established on the basis of comprehensive spectroscopic analysis, mainly using 1D and 2D NMR experiments. The benzopyrans 1 and 2 showed weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides. Analyses of the biological activities were also carried out on HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cells, aiming to evaluate their potential effects on mammalian cell line proliferation. Results from both cell lines indicated that compound 2 was able to induce cell proliferation: 70% on HeLa cells and 25% on CHO cells. © 2005 Elsevier Ltd. All rights reserved.

Isolation and growth of the symbiotic fungus of Atta capiguara (Hymenoptera: Formicidae)

This work's objectives were to isolate and evaluate the growth of the symbiotic fungus of Atta capiguara Gonçalves on artificial medium, under different pH and temperature conditions. Isolation was accomplished using the following media: Sabouraud, oat-agar, PDA, and PDA with the addition of extracts from the grasses Paspalum sp. Flügge and Hyparrhenia rufa (Nees) Stapf.. The medium used in the growth study was PDA with the addition of a Paspalum sp. (0.22%, w/v) extract at initial pH values of 4.5, 6.0, and 7.5. Mycelium disks were transferred to plates containing the culture medium. The plates were maintained at temperatures of 20, 23, and 26 ± 1°C. Mycelial radial growth evaluations were performed at 7, 14, 21, 28, and 35 days of incubation. Fungus isolation was obtained in all media studied. The highest radial means were obtained at initial pH values of 6.0 and 7.5 and temperatures of 23 and 26± 1°C. Greater plot losses occurred at the initial pH condition of 7.5. In general, A. capiguara fungi can be grown in the medium studied, at an initial pH of 6.0 and temperatures of 23 or 26± 1°C. Radial growth evaluations at 14 and 28 days of incubation can be recommended for substrate studies involving the symbiotic fungus.

Metabolism of plant polysaccharides by Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ant Atta sexdens L.

Atta sexdens L, ante feed on the Fungus they cultivate on cut leaves inside their nests. The fungus, Leucoagaricus gongylophorus, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds lay the ants, This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by L. gongylophorus, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.

Activity of Ricinus communis (Euphorbiaceae) and ricinine against the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera : Formicidae) and the symbiotic fungus Leucoagaricus gongylophorus

The focus of this study was the identification of compounds from plant extracts for use in crop protection. This paper reports on the toxic activity of fractions of leaf extracts of Ricinus communis L (Euphorbiaceae) and isolated active compounds in the leaf-cutting ant Atta sexdens rubropilosa Forel and its symbiotic fungus Leucoagaricus gongylophorus (Singer) Moller. The main compounds responsible for activity against the fungus and ant in leaf extracts of R communis were found to be fatty acids for the former and ricinine for the ants. (C) 2004 Society of Chemical Industry.

Nesting biology of the fungus growing ants Mycetarotes Emery (Attini, Formicidae)

Fungus-growing ants of the genus Mycetarotes are among the least studied in the tribe Attini. This report documents nest architecture and worker population numbers for 19 nests of M. parallelus and 5 nests of M. acutus, including the first such report for M. acutus. This new information is integrated with the scant biological information reported on Mycetarotes to date. The resulting picture of Mycetarotes life history, as well as the relative ease with which large numbers of nests can be collected and observed in the field, suggest that Mycetarotes (particularly M. parallelus) is an ideal model system for the study of coevolution of lower-attine ants and their cultivated fungi.

Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants

Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, degrades starch, this degradation being supposed to occur in the plant material which leafcutters forage to the nests, generating most of the glucose which the ants utilize for food. In the present investigation, we show that laboratory cultures of L. gongylophorus produce extracellular alpha-amylase and maltase which degrade starch to glucose, reinforcing that the ants can obtain glucose from starch through the symbiotic fungus. Glucose was found to repress a-amylase and, more severely, maltase activity, thus repressing starch degradation by L. gongylophorus, so that we hypothesize that: (1) glucose down-regulation of starch degradation also occurs in the Atta sexdens fungus garden; (2) glucose consumption from the fungus garden by A. sexdens stimutates degradation of starch from plant material by L. gongylophorus, which may represent a mechanism by which Leafcutters can control enzyme production by the symbiotic fungus. Since glucose is found in the fungus garden inside the nests, down-regulation of starch degradation by glucose is supposed to occur in the nest and play a part in the control of fungal enzyme production by leafcutters. (c) 2005 Elsevier GmbH. All rights reserved.

Synthetic amides toxic to the leaf-cutting ant Atta sexdens rubropilosa L. and its symbiotic fungus

1 Nine synthetic amides similar to natural N-piperidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenainide and N-pyrrolidine-3-(4,5-methylenedyoxiphenyl)2-(E)-propenamide were synthesized and identified by their spectroscopic data.2 the toxicity of these synthetic amides to the Atta sexdens rubropilosa workers and the antifungal activity against Leticoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, were determined.3 Workers ants that were fed daily on an artificial diet to which these compounds were added had a higher mortality rate than the controls for N-pyrrolidine-3(3',4'-methylenedioxyphenyl)-2-(E)-propenamide and N-benzyl-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at a concentration of 100 mu g/mL.4 the completely inhibition (100%) of the fungal growth was observed with N-piperldine-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide and N,N-diethyl-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at concentrations of 50 and 100 mu g/mL and N-pirrolidine-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at a concentration of 100 mu g/mL.5 the possibility of controlling these insects in the future using synthetic piperamides that can simultaneously target both organisms is discussed.