Patricia Vieira Tiago

Biological insect control using Metarhizium anisopliae: morphological, molecular, and ecological aspects

Microbial control of insects is based on the rational use of pathogens to maintain environmentally balanced pest population levels, and Metarhizium anisopliae has been the most studied and most utilized fungal species for that purpose. The natural genetic variability of entomopathogenic fungi is considered one of the principal advantages of microbial insect control. The inter- and intraspecific variability and the genetic diversity and population structures of Metarhizium and other entomopathogenic fungi have been examined using ITS-RFLP, ISSR, and ISSP molecular markers. The persistence of M. anisopliae in the soil and its possible effects on the structures of resident microbial communities must be considered when selecting isolates for biological insect control.

Isolamento e seleção de fungos filamentosos do solo de sistemas agroflorestais do Município de Bom Jardim (PE) com base na capacidade de produção de enzimas hidrolíticas

This study aimed to isolate and identify fungi from soil of agroforestry systems and to evaluate the enzymatic activity of these fungi. These were isolated by successive dilution technique and evaluated for their ability to degrade starch, cellulose and caseine. Eleven species of anamorph fungi (Hyphomycetes) distributed in five genera were identified: Aspergillus, Cladosporium, Curvularia, Fusarium and Penicillium. The species studied did not show satisfactory amylolytic activity. Cladosporium cladosporioides (Fres.) De Vries and Penicillium chrysogenum Thom had higher enzyme relation index (ERI) for protease and cellulase. Among the species studied six showed higher Relative Enzymatic Index (IRE) for cellulase in comparison to the other enzymes.

Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.

Polymorphisms in entomopathogenic fusaria based on inter simple sequence repeats

ABSTRACT Isolates of Fusarium obtained from Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae) demonstrated potential as biological control agents against that same insect, which is a pest on Opuntia ficus-indica L Miller. The isolates belong to two species complexes: Fusarium incarnatum-equiseti (FIESC – five species) and Fusarium fujikuroi (FFSC – one species). Twenty-eight isolates of these fungi were characterised using seven Inter Simple Sequence Repeats (ISSR) primers. The UBC841 primer differentiated all the FIESC isolates studied and the single isolate of Fusarium pseudocircinatum O’Donnell & Nirenberg at a level greater than 90% similarity for the fragment sizes. The results indicated high genetic variability among those isolates, an important characteristic for biological control, increasing the chances of finding efficient fungi for insect control. The ISSR markers UBC834 and UBC841 were found to be efficient for characterising and differentiating (DNA fingerprinting) those fungi, and could be used in field monitoring.

Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle

The sugarcane borer, Diatraea saccharalis , is one of the worst pests in Brazilian sugarcane crop, causing high levels of financial losses every year. Beauveria bassiana is an entomopathogenic fungus widely used in the biological control of several agricultural pests. The aims of this study were to: (1) evaluate the pathogenicity of B. bassiana strains against D. saccharalis (2) investigate the production of proteases and chitinase by B. bassiana in the presence of the cuticle of sugarcane borer; and, (3) evaluate the relation between the production of enzymes and pathogenicity of the strains. All isolates tested were pathogenic to D. saccharalis and the mortality ranged from 36 to 88%. The production of enzymes was higher in the medium containing cuticle, showing that the process is stimulated by specific components found in the cuticle of the host. Pr1 activity was higher than Pr2 and both were produced at 24 h. The highest production of chitinase was obtained at 96 h of culture for all strains tested. Levels of specific cuticle-degrading enzymes such as proteases correlated positively with specific virulence parameters. B. bassiana URM2915 showed promising features to be used in a biological control program of D. saccharalis . Key words : Biological control, sugarcane, subtilisin-like protease, trypsin-like protease, chitinase.

Dactylopius opuntiae: control by the Fusarium incarnatum–equiseti species complex and confirmation of mortality by DNA fingerprinting

Cultivation of the prickly pear cactus, Opuntia ficus-indica, an important cattle food in the semiarid region of Brazil, has been increasingly compromised by predation by the cochineal scale Dactylopius opuntiae. Entomopathogenic fungi represent a low environmental impact alternative to controlling this insect pest, and the genus Fusarium has been described as a promising agent for that task. As such, we selected isolates of the Fusarium incarnatum–equiseti species complex (FIESC) to evaluate their potential biological control of D. opuntiae and confirmed their efficiency/presence in the field through morphological and molecular characteristics. The pathogenicities of 25 isolates of FIESC obtained from D. opuntiae were evaluated. The isolates URM6782, URM6778, and URM6811 demonstrated kill rates above 45%, and good sporulation characteristics, and were thus selected for field testing. The isolate URM6782 showed the highest kill rate in the field, and amplification profiles obtained using the ISSR UBC834 marker confirmed that the isolates released in the field were in fact the causal agents of the high mortality of D. opuntiae. We also observed natural biological control by native Fusarium species present in the field. Our results suggest that the isolates URM6782 and URM6778 show significant promise for controlling D. opuntiae and that the ISSR primer UBC834 can be used to monitor those isolates when released into the field. This work represents an initial study directed toward the biological control of D. opuntiae using novel isolates from the FIESC.