Characterization of Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) isolates from Brazil and their efficacy against Aedes aegypti (Diptera, Culicidae).

AIMS: To survey and to characterize entomopathogenic fungi as natural enemies of mosquitoes in Central Brazil. METHODS AND RESULTS: Tolypocladium cylindrosporum (Hypocreales, Ophiocordycipitaceae) was isolated for the first time in South America by using Aedes aegypti (Diptera, Culicidae) as sentinel larvae in a stagnant mosquito breeding site in a secondary tropical forest. Two isolates were morphologically and molecularly identified, and their activity tested against A. aegypti eggs, larvae and adults. CONCLUSIONS: Findings emphasize the importance of T. cylindrosporum as a natural fungal antagonist of mosquitoes. SIGNIFICANCE AND IMPACT OF THE STUDY: Both isolates affected larvae and adults but were less effective against eggs; both have potential for development as a mycoinsecticide especially against larvae of A. aegypti the main vector of dengue, chikungunya, Zika and urban yellow fever.

First report of Coelomomyces santabrancae sp. nov. (Blastocladiomycetes: Blastocladiales) infecting mosquito larvae (Diptera: Culicidae) in central Brazil.

A project from 2013 to 2017 sought to discover pathogenic fungi and oomycetes from dipteran species that are vectors of major diseases of humans and animals in central Brazil and to begin evaluating the potential of these pathogens as potential biological control agents concentrated on mosquito larvae. Some collecting sites proved to be especially productive for pathogens of naturally occurring mosquito species and for placements of healthy sentinel larvae of Aedes aegypti in various sorts of containers in a gallery forest in the Santa Branca Ecoturismo Private Reserve of Natural Patrimony (RPPN) near Terezópolis de Goiás (GO). Collections during May-April of 2016 and February 2017 yielded a few dead mosquito larvae of an undetermined Onirion sp. (Culicidae: Sabethini) whose hemocoels contained many ovoid, thick-walled, yellow-golden to golden-brown, ovoid thick-walled resistant sporangia, 38.3±4×22.8±2.3µm, decorated by numerous, closely and randomly spaced punctations of variable size and shape. These were the first indisputable collections from Brazil of any Coelomomyces species. Comparisons of the morphology of these sporangia with those of other species of Coelomomyces, confirmed that this Brazilian fungus represented a new species that is described here as Coelomomyces santabrancae.

Neozygites osornensis (Neozygitales: Neozygitaceae) Infecting Cinara sp. (Hemiptera: Aphididae) in Brazil.

The genus Neozygites has been known in Brazil until now only on mites, and this is its first report on aphids in Brazil. Tree-dwelling aphids (Cinara sp.) on a cypress tree were regularly monitored for entomopathogenic fungi in the city of Terezópolis de Goiás in Central Brazil between July 2014 and April 2015. During the survey, mycosed aphids were found attached to twigs. The fungus was identified morphologically as Neozygites osornensis. No in vitro cultures were established from infected aphids. The finding reported here suggests that special attention should be paid in the future for the possible occurrences of Neozygites species wherever cinaran aphids occur around the globe.

First Report of Fusarium sporotrichioides Causing Foliar Spots on Forage Corn in Chile.

In southern Chile, forage corn (Zea mays L.) is grown for feeding animals in milk diaries and livestock production. In December 2010, corn plants with small circular spots on leaves were collected from three fields located in Río Negro (Los Lagos region). Symptoms began as small, circular white to brown spots of 5 to 10 mm on different parts of the leaf and necrotic tissue with irregular brown to burgundy margins on the border and tip of the leaf. Estimated visual severity was ~5 to 40% for each leaf from field samples. Twelve small blocks of tissue were taken from the edge of necrotic spots from infected leaves, surface disinfected (2 min in 95% ethanol, 2 min in 0.5% NaOCl, followed by three rinses with sterile distilled water), and then placed on PDA and incubated for 7 days at 24 ± 1°C. Seventy five percent of the sampled tissues developed fungal colonies and a 4-mm3 block of agar that contained the advancing hyphal edge of each colony was transferred to PDA and carnation leaf agar and incubated for 10 days at 24 ± 1°C. Colonies were fast growing with pink-white and dense mycelia; with a carmine red color on the undersurface of the plate and orange sporodochia; polyphialides abundant; microconidia abundant, oval or pear-shaped or spindle-shaped, thin walled, hyaline, often with a papilla at the base, and 5.5 to 12.2 × 2.0 to 3.2 µm. Macroconidia were sickle-shaped, 3 to 5 septate, moderately curved to straight, hyaline, thick walled, and 20.5 to 42.9 × 3.5 to 5.0 µm. Morphology of colonies and conidia matched the description of Fusarium sporotrichioides Sherb. (3). Identity of the fungus was confirmed by molecular characterization of the ITS and 18SrRNA regions (universal primers ITS4/5 and NS1/2, respectively) and the ß-tubulin gene (primers Bt1a/Bt1b) of three isolates. BLAST searches of the obtained sequences had between 99 to 100% homology with several isolates of F. sporotrichioides from GenBank (Accession Nos. KC866343 to KC866351). Pathogenicity tests were conducted by dispensing 10 µl of a prepared spore suspension (107 spores/ml) on corn leaves (16 leaves). Negative controls were corn leaves inoculated with sterile distilled water. Inoculated corn leaves were kept at 25 ± 1°C in glass bell jars and monitored for the onset of symptoms for 10 days. The test was conducted twice. Additionally, 20 corn plants of four hybrid lines were inoculated with ~5 ml of a spore suspension (104 macroconidia/ml) 2 months after seeding under field conditions in Valdivia, Los Ríos region, Chile. Seventy five days after sowing, similar lesions to those initially observed on field infected leaves were observed on inoculated leaves but not on water controls. Under field conditions, an extended damage on borders of basal leaves and spots on stems and cobs was observed. The pathogen was reisolated from infected tissues, thereby fulfilling Koch's postulates. F. sporotrichioides is a frequent pathogen in corn silage (1) and cereal crops (3,4), and produces trichothecene mycotoxins that cause toxicosis in animals (2,3). To our knowledge, this is the first report of F. sporotrichioides causing foliar spot on forage corn in Chile and this disease could represent a serious risk of mycotoxin contamination in this crop. References: (1) H. Baath et al. Arch. Tierernahr. 40:397, 1990. (2) A. E. Desjardins et al. Phytopathology 79:170, 1989. (3) J. F. Leslie and B. A. Summerell. Page 256 in: The Fusarium Laboratory Manual. Blackwell Publishing Professional, Hoboken, NJ, 2006. (4) R. H. Vargo et al. Plant Dis. 70:629, 1986.