María Guadalupe Maldonado-Blanco

The effect of oxygen tension on the production of Bacillus thuringiensis subsp. israelensis toxin active against Aedes aegypti larvae

An optimized batch production of Bacillus thuringiensis subsp. israelensis was made in a stirred Bioflo III reactor using a previously selected medium, and operating conditions in the range of 100–500 rev/min stirrer speeds and 0.2–1 air flow/culture medium volume/minute (v/v/m) aeration rates, including five combinations; at the end of fermentation, dry powders were recovered and evaluated against Aedes aegypti larvae at 0.05 mg/l. Later, the lethal concentration inducing 50% mortality (LC50) was determined for the two most toxic powders. The bioinsecticide yields varied from 9.1 to 15.7 g/l and the total fermentation times ranged between 18 and 30.3 h. The toxicity varied for two powders much more than for the others. These were for combinations with 300 rev/min:1 v/v/m and 500 rev/min:0.6 v/v/m, giving mortality percentages of 47.2 and 59.7, with LC50 values of 0.2675 and 0.0685 mg/l, respectively. A t test showed no significant difference. However, the larvicidal powder produced with 300 rev/min:1 v/v/m gave more variable toxicity than the powder obtained with 500 rev/min:0.6 v/v/m.

Predatory capacity and prey selectivity of nymphs of the dragonfly Pantala hymenaea

ABSTRACT Predatory capacity and prey selectivity of nymphs of the dragonfly Pantala hymenaea (Odonata: Libellulidae) were evaluated on larvae of the mosquito Culex quinquefasciatus (Diptera: Culicidae) and larvae of the midge Chironomus plumosus (Diptera: Chironomidae) as prey. With functional response methodology, 7 larval densities were exposed to predator individuals in a glass jar under laboratory conditions. The study was performed in 2 experiments. The 1st was a test system with each prey species alone. The 2nd tested a mixture of both prey species in a 1:1 ratio. Prey selectivity and prey capacity were significantly greater on midge larvae than on mosquito larvae.

Effect of culture medium on the production and virulence of submerged spores of Metarhizium anisopliae and Beauveria bassiana against larvae and adults of Aedes aegypti (Diptera: Culicidae)

Three Metarhizium anisopliae and three Beauveria bassiana isolates were cultivated in media containing casamino acids, soybean flour or sunflower seed flour and were shaken for three days. M. anisopliae presented similar yields of around 106 submerged spores/ml without significant differences among them, whereas B. bassiana produced yields of around 108 spores/ml, of which GHA strain produced more submerged spores in the casamino acids medium. The other two strains showed no significant difference in the production of submerged spores in the three media used. Differences in mortality on Aedes aegypti larvae were observed with the submerged spores of Metarhizium depending on isolate and medium used. M. anisopliae 2157 caused significantly higher mortality (40%) when cultivated in casamino acids medium. It presented an LC50 of 8.93 × 105 submerged spores/ml water against mosquito larvae five days after application, whereas it caused 27% mortality in Ae. aegypti adults 10 days after application. In conclusion, fungal nutrition affected virulence of some isolates of M. anisopliae against Ae. aegypti larvae while such an effect was not noted for B. bassiana isolates.

Paecilomyces fumosoroseus blastospore production using liquid culture in a bioreactor

There are many advantages to using liquid cultures for the production of blastospores. These include mainly the processes of scale up which are relatively easy, as well as the control of parameters such as temperature, aeration and pH. In this work, we evaluated the production of Paecilomyces fumosoroseus blastospores using a low-cost liquid culture medium in a fermenter in comparison to a medium commonly used for this purpose, with regard to yield and viability of blastospores. The two media contained the same concentration of glucose but differed in N source (M1 containing casamino acids and M2 provided with collagen peptone and yeast extract). Starting with an inoculum of 1x106 blastospores/ml, M2 medium produced 2x1010 blastospores/ml after incubation for 72 h at 520 rev/min agitation and 1 v/v/m (volume air/volume liquid.min) aeration, while only 2.4 x 108/ml were produced with M1. In addition, the microorganisms in medium M1 grew more slowly during log phase and reached an earlier plateau at 36 h fermentation. The medium containing collagen peptone and yeast extract is an excellent alternative for the production of P. fumosoroseus blastospores, providing lower cost, higher yield and shorter propagation time, but formulation does need to be improved.

Radial Growth, Sporulation, and Virulence of Mexican Isolates of Hirsutella citriformis1 against Diaphorina citri2

Abstract. Hirsutella citriformis Speare is an important entomopathogenic fungus that infects diverse species of hemipteran insects, including the vector of huanglongbing, Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). The objective of this study was to determine the growth and conidiation of eight isolates of H. citriformis as well as virulence of the conidia on Asian citrus psyllid adults and nymphs. The strains were isolated from infected D. citri in states of Mexico where citrus, Citrus spp., are grown. Optimum radial growth was obtained at 25°C for the eight isolates incubated in three agar media (potatodextrose agar, potato dextrose agar with 0.5% yeast extract, and Sabouraud dextrose agar). Most daily radial growth was by six strains and most production of conidia (1.833 × 107 per milliliter) was by strain IB-Hir-2 after 34 days of incubation on potato dextrose agar with 0.5% yeast extract. In a preliminary bioassay, strains INIFAP-Hir-1, IB-Hir-2, and IB-Hir-1 killed most (88.1, 87.7, and 85.6%, respectively) Asian citrus psyllid adults inoculated by contact. From 50.5–81.7% of the nymphs died.

Mexican strains of Hirsutella isolated from Diaphorina citri (Hemiptera: Liviidae): Morphologic and molecular characterization

Abstract Diaphorina citri Kuwayama (Hemiptera: Liviidae), the vector of huanglongbing, has been found in several citrus areas of Mexico, apparently attacked by the fungus Hirsutella citriformis Speare (Hypocreales: Ophiocordycipitaceae). As this entomopathogen could represent a potential agent of D. citri biological control in the country, we performed this study to characterize 7 strains of the fungus isolated from this insect. For the molecular characterization we used internal transcribed spacer sequences 1 and 2 (ITSs 1 and 2) and 28S rDNA. In the morphological description we determined size of reproductive structures in the strains as well as their blastospore production capacity. BLAST analysis of the ITSs sequences showed less than 91% and 72% of identity and coverage with Ophiocordyceps sinensis or H. citriformis; meanwhile, gen 28S sequences showed high identity (98–99%) and coverage (98–100%) with O. elongata (Petch) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora and O. coccidiicola (Kobayasi) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. The phylogenetic analysis using 28S sequences demonstrated that the Mexican strains are closely related with H. citriformis; however, size of phialide and conidial diameter showed differences with dimensions reported for the species in the original description. Under dark conditions, 6 strains were able to produce mucilaginous colonies that contained blastospores; these structures caused 30.8–41.2% mortality in the target insect. The results suggest that Hirsutella strains isolated from D. citri in Mexico, correspond to H. citriformis. We found dimorphic capacity and variability in size of the reproductive structures of the fungus. This is the first report of ITSs and 28S sequences of H. citriformis obtained from strains isolated from D. citri

Effects of culture medium and formulation on the larvicidal activity of the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) against Aedes aegypti

In this work, we examined the production of infective zoospores of Lagenidium giganteum in four culture media, and the larvicidal activity of the cultures was determined against Aedes aegypti larvae, as well as the effect of polymer encapsulation. Medium containing sunflower seed extract showed the greatest production of zoospores, 5.92×10(6) zoospores/ml after six days of fermentation at 25±2°C and 150rpm shaking. This culture tested against A. aegypti 1st stage larvae caused different mortality rates at 24, 48 and 72h posttreatment. The LC(50) obtained was 43.9, 41.1 and 42.9μl of total culture/ml, at 24, 48 and 72h posttreatment respectively, while the culture grown in medium with soybean meal showed 3-5 times higher LC(50) values. Finally, the total culture including mycelium, zoospores and presporangia formulated with 2.5% pectin showed significantly higher mortality rates, around 100% more than the unformulated culture, whose values were from 40 to 1% at 3, 6, 9, and 12d posttreatment in the bioassays carried out in the laboratory to determine residual activity.

Evaluation of Mexican Strains of Hirsutella citriformis1 Against Diaphorina citri2 in a Semifield Bioassay

Abstract. Citrus production is threatened by Huanglongbing disease transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama. Developing strategies to control the vector is necessary. Conidial infectivity of four Mexican strains of Hirsutella citriformis Speare (INIFAP-Hir-2, INIFAP-Hir-4, IB-Hir-1, and IB-Hir-2) was evaluated in a semifield bioassay against adult Asian citrus psyllid. The bioassay was done in an orchard of Persian lime (Citrus aurantiifolia (Christm. et Panz.) Swingle var. latifolia Yu. Tanaka) at Veracruz, Mexico. The four strains showed pathogenicity, but INIFAP-Hir-2 and INIFAP-Hir-4 killed most (51.05-50.6%), with no significant difference between them. Thirty days after application in the field, several H. citriformis strains were recovered from mycosed insects and grown in different media where they varied depending on the culture medium and strain and presented morphological characteristics similar to the strains applied and to a local strain previously isolated. Mycosed insects increased from 2.3 to 10.0 per tree 30 days after application of strains of H. citriformis in the orchard. The four H. citriformis strains applied by spraying showed pathogenicity against the Asian citrus psyllid under semifield conditions. Entomopathogenic fungi such as H. citriformis could be a promising alternative in an integrated management program against Huanglongbing, but need more evaluation in the field.

Effect of Mexican Hirsutella citriformis (Hypocreales: Ophiocordycipitaceae) Strains on Diaphorina citri (Hemiptera: Liviidae) and the Predators Chrysoperla rufilabris (Neuroptera: Chrysopidae) and Hippodamia convergens (Coleoptera: Coccinellidae)

Abstract Due to its role in the transmission of Candidatus Liberibacter asiaticus, a pathogen associated with huanglongbing, a catastrophic disease of citrus in the world, Diaphorina citri Kuwayama (Hemiptera: Liviidae) has become a very dangerous invasive pest. To contribute to increasing alternatives for its management, we evaluated against D. citri adults the pathogenicity of conidia and blastospores of 8 Mexican strains of the entomopathogenic fungus Hirsutella citriformis Speare (Hypocreales: Ophiocordycipitaceae). Furthermore, we conducted tests with non-target insects that included the predators Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) and Chrysoperla rufilabris (Burmeister) (Neuroptera: Chrysopidae). Experiments in the laboratory included the application of the fungus by contact and through liquid sprays. Strains were collected from citrus areas of Mexico. They were grown in PDAY media and applied at 1 × 106 conidia per mL. All bioassays were conducted under controlled conditions at 26 ± 1 °C, a 16:8 h L:D photoperiod, and 76 ± 4% RH and maintained during 26 d after inoculation. Tests with D. citri were performed 3 times at different dates. Mean mortality by H. citriformis strains on D. citri adults ranged from 82 to 92%; INIFAP-Hir-1 strain produced the highest rate. Sprayed conidia produced 69% mortality. Use of sprayed blastospores caused 32 to 49 % mortality. LC50 obtained for INIFAP-Hir-1 strain was 3.4 × 106 conidia per mL. IB-Hir-1, IB-Hir-2, INIFAP-Hir-1, and INIFAP-Hir-2 strains inoculated by contact on H. convergens adults caused 9 to 11% mortality; in larvae of C. rufilabris, mortality ranged from 19 to 25%. In both tests, there were no statistical differences when compared with the untreated control. Unlike D. citri, all dead predator specimens showed absence of H. citriformis mycosis. The results suggest potential for the integration of this fungus in the management of D. citri. Data obtained from the predators could support safe use of this biological control agent.

Conidial Production on Different Solid Substrates and Pathogenicity of Hirsutella citriformis1 Against Diaphorina citri2 Adults

Abstract. Conidia of four strains of H. citriformis Speare fungi grown on oat (Avena sativa L.), wheat (Triticum aestivum L.), and sorghum (Sorghum bicolor (L.) Moench) were evaluated for production and for viability and pathogenicity against D. citri Kuwayama adults. Substrates were inoculated with conidial suspensions (1 × 106 conidia/ml) of INIFAP-Hir-1, INIFAP-Hir-2, IB-Hir-1, and IB-Hir-2 strains and incubated for 35 days. Pathogenicity on Asian citrus psyllid adults also was evaluated through contact bioassay. Significantly more conidia were produced on oat (58.5-28.83 × 106 per gram) than wheat (50.9-29.4 × 106 per gram) at 21 days. Production of most conidia (8.4-6.6 × 106 per gram) required 28 days on sorghum and was less than on oat or wheat. Percentages of germination of conidia on the three substrates ranged from 96.25 to 98.0% without significant differences among them. Pathogenicity of the strains against Asian citrus psyllid were not significantly different when grown on agar (85.5–89.9%) or oat (85.8–89.3%). Results confirmed the possibility of increased production of H. citriformis conidia in natural substrates maintaining pathogenicity against Asian citrus psyllid.