RESUMO

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

Assuntos

Antibiose/fisiologia , Bacillus subtilis/fisiologia , Bactérias/crescimento & desenvolvimento , Fungos/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Fragaria/microbiologia , Fusarium/crescimento & desenvolvimento , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , México , Peptídeo Hidrolases/metabolismo , Peptídeos/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase , Rhizoctonia/crescimento & desenvolvimento

RESUMO

In 2008 and 2009, vine decline symptoms were observed in three watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) fields located in the municipalities of Mossoró (Rio Grande do Norte State) and Quixeré (Ceará State) in northeastern Brazil. Symptoms included yellowing of crown leaves just prior to harvest and collapse of many of the vines. Mean maximum daily temperatures for the first and second half of the season were 28.6 and 25.1°C, respectively. Affected plants exhibited necrotic root systems and lacked most of the secondary and tertiary feeder roots. Numerous perithecia on the roots contained asci and ascospores characteristics of Monosporascus cannonballus Pollack & Uecker (1,2). Small pieces of primary and secondary roots were surface disinfected and plated onto potato dextrose agar (PDA) medium with 0.5 g liter-1 of streptomycin sulfate and incubated for 7 days at 25°C in the dark. Hyphal tips from all colonies were transferred to PDA and further incubated for 30 to 40 days at 25°C in the dark for subsequent growth and sporulation. Isolations consistently yielded colonies of white mycelium, which became dark grayish after 10 to 15 days, and perithecia with one-spored asci. The internal transcribed spacer regions of ribosomal DNA of isolates 18-5 and 19-1 were sequenced (GenBank Accession Nos. GQ891544 and GQ891545). These sequences were identical to sequences of M. cannonballus (GenBank Accession Nos. AM167936 and AM167937). Pathogenicity of these two isolates was confirmed on watermelon cv. Crimson Sweet in a greenhouse maintained at 25 to 30°C. Inoculum was produced in a sand-oat hulls (Avena sativa) medium (0.5 liter of sand, 46 g of ground oat hulls, and 37.5 ml of distilled water) and incubated at 25°C for 1 month. CFU were quantified by serial dilution using 1% hydroxyethyl cellulose. A sterilized mixture of equal portions (vol/vol) of sand and peat moss was used to fill 17-cm-diameter plastic pots and inoculum was added to produce an inoculum concentration of 20 CFU g-1. Five watermelon seeds planted in each pot were later thinned to one seedling per pot. There were five replicated pots for each treatment with an equal number of noninfested pots. Plants were evaluated for disease 45 days after sowing. All isolates of M. cannonballus were highly aggressive and caused severe root necrosis compared with the noninoculated controls. M. cannonballus was reisolated from symptomatic plants, confirming Koch's postulates. In 2004, M. cannonballus was reported in the same Brazilian cucurbit-growing areas causing root rot and vine decline of muskmelon (Cucumis melo L.) (3), but to our knowledge, this is the first report of M. cannonballus on watermelon in Brazil. References: (1) R. D. Martyn and M. E. Miller. Plant Dis. 80:716, 1996. (2) F. G. Pollack and F. A. Uecker. Mycologia 66:346, 1974. (3) R. Sales Jr. et al. Plant Dis. 88:84, 2004.

RESUMO

Mesquite (Prosopis pallida (Wildenow) Kunth) is a drought-tolerant tree widely distributed in the northern Pacific Coast of South America. This species prevents soil erosion, provides shade, conserves prairies, supports bee nutrition, and provides fruits for human and animal consumption. Since the spring of 2004, bark lesions and bleeding cankers were observed on trunks and branches of 70% of declining mesquite trees in some parks at Ica in southern Peru. Badly affected trees were killed by the disease. Isolations were made from the edge of necrotic lesions of the inner bark and roots using PARPH medium (2) and incubated at 22°C for 7 days. A Phytophthora species was consistently isolated from lesions of 10 mesquite trees, and six pure cultures (PS-87-PS-92) were obtained by transferring hyphal tips and characterized. Colonies were stellate on V8 juice agar (VJA; 2 g CaCO3, 200 ml of V8 juice, and 15 g of agar in 800 ml of distilled water), uniform to slightly radiate on corn meal agar (Oxoid Ltd., London, England), and knotty on PDA (Biokar Diagnostics, Beauvais, France). On VJA at 22°C, the average radial growth rate for the six isolates was 1.7 mm per day. Colonies grew slowly at 5 and 25°C with 0.4 and 0.7 mm per day growth rate, respectively. There was no growth at 30°C. Catenulate hyphal swellings formed on VJA and liquid media (1.5% sterile soil extract). Sporangia were persistent, ovoid to obpyriform, semipapillate with narrow exit pores (<5.0 µm in diameter), 32.3 to 39.7 × 21.0 to 27.2 µm, with a length/width ratio of 1.4:1 to 1.6:1. Sporangia were produced by cutting 5-mm disks from the advancing margin of a colony on VJA and adding disks to 10 ml of 1.5% sterile soil extract for 4 to 5 days at 22°C under fluorescent light. Isolates were homothallic with spherical oogonia, 32 to 35 µm in width with paragynous antheridia, and aplerotic oospores, 26 to 31 µm. These characteristics fit the descriptions of Phytophthora syringae (Kleb.) Kleb. (1). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. syringae (Accession No. AJ854297 from Citrus limon). In 2005, two methods were used to inoculate mesquite with two isolates. One method used two 20-mm-diameter branches of five 5-year-old mesquite trees where a 5-mm wound was made with a cork borer and a 5-mm block of the agar culture was placed under the bark and sealed with Parafilm. Another method used 10 4-month-old potted plants that received a 30-ml drench of a 104 zoospores/ml suspension per plant. Controls received clean agar blocks and a sterile water drench for 10 control pots. Two weeks after inoculation, black areas and resinosis were observed around inoculated wounds. Inoculated branches produced cankers of 4.7 to 6.8 cm2, 4 weeks after inoculations. Twenty days after inoculation of roots, wilting and root rots of seedlings occurred. No symptoms were found on the control plants. P. syringae was reisolated from the diseased branches and root rots and pure cultures were established. This test was repeated for both methods with similar results. To our knowledge, this is the first report of P. syringae in Peru and the first description of this pathogen on mesquite worldwide. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (2) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.

RESUMO

Approximately 15,000 ha of melon (Cucumis melo L.) are grown in the northeastern section of Brazil, mostly for export to Europe during the winter months. Surveys for melon vine decline diseases were carried out in farms in the municipalities of Mossoró (Rio Grande do Norte) and Quixeré (Ceará) during 2002 and 2003. Symptoms typical of vine decline were observed in several fields and included yellowing of crown leaves just prior to harvest and collapse of many of the vines. Affected plants exhibited necrotic root systems and lacked most of the secondary and tertiary feeder roots. Numerous perithecia were observed on roots which, when examined with a microscope, showed characteristic asci and ascospores of the fungus Monosporascus cannonballus Pollack & Uecker (2). Isolations were made from the crown region and primary and secondary roots of affected plants by excising 4- to 6-mm pieces that were surface sterilized for 30 to 60 s with 1.5% active chlorine solution. Seven tissue pieces from each plant part were placed on potato dextrose agar (PDA) containing 0.5 g liter-1 of streptomycin sulfate. Plates were examined daily for fungal growth for 7 days, and hyphal tips from all colonies were transferred to PDA for subsequent growth and sporulation. M. cannonballus was isolated from 50% of the root sections. All isolates produced only one ascospore per ascus. Pathogenicity of four isolates was confirmed in the greenhouse on the muskmelon cv. Temprano Rochet. Inoculum was produced in a sand-oat hulls (Avena sativa L.) medium (0.5 liter of sand, 46 g of ground oat hulls, and 37.5 ml of distilled water) and incubated at 25°C for 1 month. Colony forming units (CFU) were quantified by serial dilution using 1% hydroxyethyl cellulose. A sterilized mixture of equal portions (vol/vol) of sand and peat moss was used to fill plastic pots (17 cm in diameter), and inoculum was added to produce an inoculum concentration of 20 CFU g-1. Five melon seeds were planted in each pot and after germination, were thinned to one seedling per pot. There were five replicated pots for each treatment with an equal number of uninfested pots. Plants were evaluated for disease 45 days after sowing. Roots were exposed by carefully washing the potting mix away. All isolates of M. cannonballus tested were highly aggressive and caused severe root necrosis compared with the noninoculated control plants. M. cannonballus was reisolated from symptomatic plants, confirming Koch's postulates. Double cropping in the same fields for several years has created serious problems in Brazil, which are related to this soilborne pathogen that also causes root rot and vine decline of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) worldwide (1). To our knowledge this is the first report of M. cannonballus in Brazil and South America. References: (1) R. D. Martyn and M. E. Miller. Plant Dis. 80:716, 1996. (2) F. G. Pollack and F. A. Uecker. Mycologia 66:346, 1974.