Detection and kinetic analysis of Epinotia aporema granulovirus in its lepidopteran host by real-time PCR.

Epinotia aporema granulovirus (EpapGV) has attracted interest as a potential biocontrol agent of the soybean pest Epinotia aporema in Argentina. Studies on virus/host interactions conducted so far have lacked an accurate method to assess the progress of virus load during the infection process. The present paper reports the development of a real-time PCR for EpapGV and its application to describe viral kinetics following ingestion of two different virus doses by last-instar E. aporema larvae. Real-time PCR was shown to be a reliable method to detect and quantify the presence of EpapGV in the analyzed samples. The increase in virus titer (log) exhibited a sigmoidal pattern, with an exponential growth phase between 24 and 48 h postinfection for both initial doses tested.

Glomerella Leaf Spot Caused by a Nonhomothallic Strain of Glomerella cingulata on Highbush Blueberry Nursery Plants in Buenos Aires, Argentina.

In February 2009, irregular-shaped leaf spots affected blueberry (Vaccinium corymbosum L. 'Blue Crisp', 'Misty', and 'Sharp Blue') nursery plants in Buenos Aires. Single-spore cultures on potato dextrose agar and oat agar showed aerial white mycelium that turned light and dark gray, dark brown acervuli with setae, and a salmon-to-orange conidial mass. Septate, dark brown, 62 to 78 µm long setae were abundant in the acervulus. Conidia were unicellular, hyaline, straight, cylindrical, round at the ends, and averaged 15.2 (12.1 to 16.9) × 5.4 (4.9 to 6.2) µm. Dark brown, ovate to clavate, 10.25 × 6.25 µm (9 to 12 × 5 to 8) appressoria with a noticeable pore formed on slides near the edge of the cover glass. Dark subglobose structures were recorded immersed in the culture medium. No asci or ascospores were observed, indicating a nonhomothallic condition. The fungus was identified as Colletotrichum gloeosporioides (Penz.) Penz & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld. & H. Schrenk) with traits similar to those already described (1). DNA was obtained from mycelium with a standard DNA extraction kit and the ribosomal, internal transcribed spacer (ITS) 1 and ITS2 regions were PCR amplified and sequenced with primers ITS1 and ITS4 (2). A BLASTN algorithm search revealed 100% identity of the sequence (535 bp long) with G. cingulata/C. gloeosporioides from citrus and mango and one from coffee identified as C. kahawae (GenBank Accession No. JF908919). The nucleotide sequence was deposited in GenBank (Accession No. JQ340087). Pathogenicity was verified on young plants and detached leaves of highbush blueberry 'Emerald', 'Misty', 'O'Neal', and 'Santa Fe', olive (Olea europaea 'Arbequina'), and marketed fruits of apple, mango, orange, and tomato. Disinfected healthy leaves were inoculated with a 9-mm2 mycelial block and incubated at 24°C with 12 h of light. Young plants were infected by placing the disinfected end of the branches within a micropipette tip filled with mycelium and kept under greenhouse conditions. Asymptomatic fruits of apple, mango, orange, and tomato were inoculated by placing a mycelial block on a small wound made on their surface. Detached leaves of highbush blueberry 'Emerald', 'O'Neal', 'Misty', and 'Santa Fe' showed 0.1 to 1.5 × 0.8 to 2 cm necrotic lesions after 3 days, covering 43 to 100% of the 'Emerald' leaf area after 8 days. Young plants of blueberry 'Emerald' and 'Misty' showed 1.5 to 3 cm necrotic lesions, acervuli, a salmon-orange conidial mass, and death of leaves at 25 days. On olive 'Arbequina', leaf necrotic lesions reached 0.1 to 3.5 cm after 5 days. Symptoms developed slowly on infected tomato fruits while inoculated fruits of apple, mango, and orange showed dark brown lesions that measured 2 to 7 × 1 to 3.5 cm at 5 days. No symptoms were observed on controls. The fungus was reisolated from inoculated plant parts. The disease was previously cited in Argentina (3), but to our knowledge, this is the first report of a nonhomothallic strain of G. cingulata from highbush blueberry colonizing and deteriorating fruits of apple, mango, orange, and tomato. References: (1) J. M. E. Mourde. No 315. CMI Descriptions of Pathogenic Fungi and Bacteria. Kew, Surrey, UK, 1971. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) E.R. Wright et al. OEPP/EPPO Bull. 28:219, 1998.

First Report of Root Rot Caused by Fusarium proliferatum on Blueberry in Argentina.

In 2009, a highbush blueberry (Vaccinium corymbosum L. 'O'Neal') field located in Rojas, Buenos Aires Province showed 30% of plants with dry or dead branches. Disinfected root pieces were placed on water agar and incubated at 24°C. A fungal colony was obtained and purified by successive transfers of an individual hyphal tip from a sparsely growing colony. Colony color and growth rate were evaluated in potato dextrose agar where the fungus produced white-to-pale pink colonies and grew 3.5 cm after 5 days. The fungus was studied on Spezieller Nährstoffarmer agar (2), carnation leaf-piece agar, and KCl agar where it produced abundant single-celled hyaline microconidia in moderate-length chains and in false heads originated from monophialides and polyphialides. Microconidia measured 6 to 12 × 2 to 3 µm (average 8 × 2.3 µm). On KCl, chains of microconidia and tan-to-light cream sporodochia with 3- to 5-septate, slender, relatively straight macroconidia were easily observed after 4 and 10 days, respectively. Macroconidia measured 38 to 48 × 3.5 to 4 µm (average 43.9 × 3.9 µm). Chlamydospores and sclerotia were not present. Data coincided with the description for Fusarium proliferatum (Matsush.) Niremberg ex Gerlach & Niremberg. The isolate was deposited in the IMYZA Microbial Collection as INTA-IMC 144. The fungus was cultured in 100 ml of Czapek-Dox supplemented with sucrose, peptone, yeast extract, sodium nitrate, and vitamins for 4 days. Genomic DNA was obtained with a DNA extraction kit, PCR amplified with primers ITS1 and ITS4 for the internal transcribed spacer (ITS) region of ribosomal genes, and sequenced. The nucleotide sequence (Accession No JF913468) was compared with GenBank records. The sequence shared 99% identity with Accession No HQ113948 for F. proliferatum. Pathogenicity was confirmed in 1-year-old 'O'Neal' plants. A 10-ml suspension (2.4 × 106 conidia/ml in sterile distilled water) was applied to six potted plants grown in sterilized potting mix. Roots were superficially wounded with a needle. Control plants were treated with sterile distilled water. Plants were incubated at 24°C and a 12-h photoperiod. After 90 days, plants showed root rot, leaf chlorosis, and branch necrosis followed by plant death. Control plants remained healthy. F. proliferatum was reisolated from diseased roots of inoculated plants. This fungus was previously cited in Argentina on asparagus (1), corn (1,3), and oat (4). To our knowledge, this is the first report of F. proliferatum as a root pathogen of highbush blueberry in Argentina. References: (1) G. Lori et al. Plant Dis. 82:1405, 1998. (2) H. I. Nirenberg. Releases Fed. Biol. Res. Ctr. Agric. For. (Berlin-Dahlem) 169:1, 1976. (3) D. A. Sampietro et al. Fung. Biol. 114:74, 2010. (4) S. A. Stenglein et al. Plant Dis. 94:783, 2010.

First Report of Fusarium solani Causing Root Rot of Olive in Southeastern Argentina.

In Argentina, olive (Olea europaea L.) is cultivated in the mountainous, warm, arid northwest (Andes range), where Fusarium solani (blue sporodochia) is frequently found to be causing death of nursery and young field plants (1). Recently, olive orchards were established more than 1,600 km to the southeast (Pampas) in a plain with a temperate and humid climate and in the arid Patagonia, both influenced by the Atlantic Ocean. This area includes part of Buenos Aires and Rio Negro provinces. In March 2008, 10-year-old 'Barnea' olive trees with high incidence of root rot, dried leaves, dead branches, and dead plants were observed in the Coronel Dorrego District of Buenos Aires Province, where oat, barley or other cereals are planted between rows of olive trees. Planting material originated from olive nurseries located in Mendoza Province, 1,200 km from Coronel Dorrego. Diseased roots were disinfected in 2% NaOCl and 70% ethanol, cut into small pieces, plated onto rose bengal-glycerin-urea medium, and incubated at 20°C with a 12-h photopheriod. A fungus was purified through successive transfers of hyphal tips from the margin of a sparsely growing colony onto 2% water agar (2). Colonies grown on Spezieller Nährstoffarmer agar (3) and carnation leaf-piece agar were used for morphological identification, and those on grown on potato dextrose agar were used for evaluation of pigmentation and colony growth rate. Sporodochium color, cream, was typical of F. solani (Mart.) Sacc. This isolate was deposited in the IMYZA Microbial Collection as INTA-IMC 73. Mycelium was cultured in liquid Czapek-Dox medium supplemented with sucrose, peptone, yeast extract, sodium nitrate, and vitamins for 4 days and fungal DNA was obtained with a DNA extraction kit. Primers ITS1 and ITS4 were used to amplify the internal transcribed spacer (ITS) region of ribosomal genes. The purified PCR product was sequenced and the DNA sequence compared with GenBank records. The sequence shared 100% identity with 27 entries for F. solani and 97% identity with F. solani obtained from olive in Nepal (4), corresponding to EU912432 and EU912433. The nucleotide sequence was registered in GenBank as JF299258. Pathogenicity was confirmed on 'Manzanilla' plants at the eight-leaf stage. Pieces of water agar with mycelium were applied to small wounds at the stem base and on roots of 10 plants and were covered with cotton soaked in sterile distilled water. Plants were incubated at 20°C and a 14-h photoperiod. On control plants, water agar pieces without mycelium were applied to the wounds. After 33 days, inoculated plants showed dark brown lesions (average length 1.4 cm) and leaf chlorosis. Two plants showed wilting with leaves remaining attached to branches. F. solani was reisolated from roots and stem bases of inoculated plants. Controls remained asymptomatic. To our knowledge, this is the first report of F. solani occurring on olive in the temperate part of the Pampas of Argentina where cereals, which are susceptible to Fusarium species, are grown with olive trees. Sporodochium color (cream) of these isolates differed from the blue color of previously reported isolates of F. solani on olive in northwestern Argentina (1). References: (1) S. Babbitt et al. Plant Dis. 86:326, 2002. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) H. I. Nirenberg. Releases Fed. Biol. Res. Center Agric. For. (Berlin-Dahlem) 169:1, 1976. (4) A. M. Vettraino et al. Plant Dis. 23:200, 2009.

First Report of Sclerotinia Rot on Blueberry Caused by Sclerotinia sclerotiorum in Argentina.

In October 2007, blighted shoots were observed on highbush blueberry (Vaccinium corymbosum L. cv. O'Neal) plants in La Plata, Buenos Aires Province, Argentina. Isolations from surface-disinfested shoots onto carrot agar and Spezieller Nahrstoffarmer Agar (SNA) consistently yielded white colonies that produced black sclerotia, mainly near the edge of the culture plates, after 7 days. Sclerotia were transferred to SNA tubes and kept at 5°C for several months. The germination of sclerotia produced numerous 6 mm long initials, stipitate pale brown cup-shaped apothecia (10 × 6 mm) with eight-spored asci (137 × 7 µm) at 18°C and continuous light conditions. Asci with uniseriate ascospores were cylindrical and narrow at the base. Ascospores (11 to 12 × 4 µm) were hyaline, unicellular, smooth, and ellipsoid. This isolated fungus was morphologically identified as Sclerotinia sclerotiorum (Lib.) de Bary (2,3). The isolate was deposited in the IMYZA Microbial Collection as INTA-IMC 87. Mycelium was cultured in 100 ml of Czapek's-Dox medium, supplemented with sucrose, peptone, yeast extract, sodium nitrate, and vitamins (1), for 3 days and fungal DNA was obtained using a DNA extraction kit. ITS1 and ITS2 of ribosomal genes were amplified by PCR using universal primers (4) and the PCR product was sequenced. A BLAST algorithm search revealed 100% identity of the sequence with 12 GenBank entries for S. sclerotiorum. The nucleotide sequence was deposited in the GenBank with Accession No. JF277567. Pathogenicity testing was achieved by placing detached leaves of cvs. Emerald, Misty, and Start on water agar (WA) plates, inoculating with 9-mm2 mycelial blocks, and incubating at 20°C with 12 h of light. Young shoots of highbush blueberry, Misty and O'Neal, were inoculated by the cut shoot method with micropipette tips filled with mycelium and kept under greenhouse conditions at 24°C and 14 h of light. On control plants, WA blocks or WA-filled micropipette tips were used. Leaf blight was observed after 5 to 6 days and sclerotia appeared after 7 days on inoculated tissues. Shoot blight was recorded on inoculated plants after 5 days. The fungus was reisolated from inoculated tissues, with no symptoms showing on controls. To our knowledge, this is the first report of Sclerotinia rot caused by S. sclerotiorum in blueberry in Argentina. References: (1) J.F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Hoboken, NJ, 2006. (2). J. E. M. Mourde and P. Holliday. No. 513 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Kew, Surrey, UK, 1976. (3) S. Umemoto et al. Gen. Plant Pathol. 73:290, 2007. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Evaluation of Beauveria bassiana (hyphomycetes) strains as potential agents for control of Triatoma infestans (Hemiptera: Reduviidae).

Chagas disease constitutes a major human health problem in most Latin American countries. This endemic disease is transmitted by several species of triatomine bugs, the most important being Triatoma infestans (Klug). In this article, we report on the selection of strains of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. virulent to T. infestans for possible use as bioinsecticides. Four strains of B. hassiana isolated from Argentina (Bb 1, 10, 25, and 65) were evaluated. To calculate mortality and mean lethal time, nymphs and adults of T. infestans were treated with conidia produced on complete agar medium and wheat brain and rice husk medium (WH). The LD50 for nymphs and adults of T. infestans was calculated. The effect of different temperatures (18, 22, 26, 30, and 34 degrees C) and relative humidities (35 and 90% RH) on mortality of nymphs were studied. We evaluated the compatibility of strains with Deltamethrin and Beta-Cypermethrin. Although the strain Bb 25 failed to grow on WH, the other three strains showed similar mortality values independent of the culture medium used to grow the microorganisms. The lowest LD50 values for nymphs were obtained with the strains Bb 10 and 65 and for adults were Bb 1, 10, and 65. The highest mean mortality was obtained with strain Bb 10, and among temperatures the highest mean mortality was observed at 26 degrees C. Relative humidity did not affect the mortality of T. infestans nymphs with all strains and temperatures assayed. Deltamethrin did not affect any of the three strains for the four studied doses, and Beta-Cypermethrin could be used in combination with the fungus only at low doses. The strain Bb 10 was selected for future assays under natural climatic conditions.