Isela Quintero-Zapata

Optimization of Pathogenicity Tests for Selection of Native Isolates of Entomopathogenic Fungi Isolated from Citrusgrowing Areas of México on Adults of Diaphorina citri Kuwayama (Hemiptera: Liviidae)

ABSTRACT Huanglongbing (HLB), considered one of the most lethal diseases of citrus worldwide, has reached the main areas of Mexican lime (Citrus latifolia Tanaka) fruit production on the Pacific coast of México. Growers have initiated intensive use of insecticides in order to control populations of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), the vector of the pathogen, ‘Candidatus Liberibacter asiaticus’ associated with huanglongbing. Presently, costs of insecticides and the side effects of their use are major concerns, because they could impair the management strategy against the vector; and thus, ecologically and economically viable alternatives to conventional insecticides are required in the short term. Therefore the goal of this study was to evaluate the pathogenicity of 27 native isolates and 3 strains of entomopathogenic fungi and determine their potential as biological control agents of D. citri by using 2 different bioassay methods. Bioassays were performed under laboratory conditions (26 ±2 °C, 60 ±5% RH and 16:8 h L:D) by exposing adult insects to a concentration of 1 × 108 conidia per milliliter using 2 different application methods, i.e., spraying the spores onto the citrus seedlings and spraying the spores directly onto the adult psyllids. The results showed that by direct spraying the adults, HIB-24 (B. bassiana) and HIB-32 (I. fumosorosea) isolates showed the highest mortality (60.66%). Regarding spraying of the seedlings, HIB-19 (I. fumosorosea) showed the highest percentage of mortality (62.02%). The results from this study demonstrate potential for using entomopathogenic fungi in the management of D. citri in México.

Isolation and Characterization of Entomopathogenic Fungi Obtained from Citrus-Growing Areas of Mexico

Abstract. A total of 142 soil samples was collected from different Mexican states: Campeche (13), Michoacán (15), Nuevo León (25), San Luis Potosí (25), Sinaloa (17), Sonora (17), Tabasco (5), Tamaulipas (20), and Yucatán (5). Larvae of the greater wax moth, Galleria mellonella (L.), were used as bait to detect, trap, and multiply entomopathogenic fungi in vivo. Twenty-three percent of the soil samples processed were positive for the presence of entomopathogenic fungi according to the macroscopic and microscopic characteristics: Beauveria bassiana (Bals.-Criv.) Vuill. was detected in 12% (17 isolates), Metarhizium anisopliae (Metchnikoff) Sorokin in 1% (2 isolates), and Isaria fumosorosea (Wize) in 10% (14 isolates). Genetic variability of the fungi was analyzed using the sequences of internal transcribed ribosomal genes (ITS-5.8S-ITS2) and digested with the enzyme Haelll. PCR results generated fragments of 600 bp for B. bassiana and M. anisopliae, and more than 600 bp for I. fumosorosea. Both reference strains and field isolates produced similar restriction patterns. Genera of native entomopathogenic fungi were found in the diverse citrus areas of Mexico, opening the possibility for biological control of pests affecting citrus in each region.

Patogenicity of Native Entomopathogenic Fungi from the Mexican Citrus-Growing Area against Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Abstract. The aim of this study was to evaluate the pathogenicity and virulence of 16 native isolates and three collection strains of three species of entomopathogenic fungi that may have the potential for affecting Asian citrus psyllid nymphs, Diaphorina citri Kuwayama (Hemiptera: Liviidae), by developing an effective methodology for their evaluation. Bioassays were conducted in a laboratory using shoots of Citrus aurantium L., infested with second to fifth instar D. citri nymphs placed in Petri dishes and sprayed with 1 × 108 conidia mL-1 suspensions of the entomopathogenic fungi Beauveria bassiana, Isaria fumosorosea, and Metarhizium brunneum. Infested insects were incubated in a growth chamber at 26 ± 2°C, 60 ± 5% relative humidity, and 16:8 lightdark hours. The results showed that /. fumosorosea Pfr-612 strain scored the highest mortality (84.2%), while the isolate HIB-14 of B. bassiana induced the development of mycosis more effectively on psyllid nymphs.

Evaluation of conidia production and mycelial growth in solid culture media from native strains of entomopathogenic fungi isolated from citrus-growing areas of México

It is important to know the ability of native strains to carry out the process of sporulation and growth in different conditions and to determine their possible potential as biological control of pests of agricultural importance, mainly in citrus areas. The objective of this study was to evaluate five different solid culture media for the determination of the production of conidia and mycelial growth of twenty-five native isolates from the Mexican states of Sinaloa, San Luis Potosi, Nuevo Leon and Tamaulipas, and three collection strains using five different solid culture media. The results showed no statistical significant difference between the five media tested for the production of conidia per milliliter and mycelial growth among different isolates. However between isolates a significant difference was found. For conidia per milliliter, the isolate with the highest production of conidia was HIB-4 with an average of 4.85 x 10 9 (spores/ml). With respect to mycelial growth strain Ma presented the highest value with 8.06 cm on average. Key words : Entomopathogenic fungi, esporulation, conidia, mycelial growth.

Effect of Native Mexican Isolates of Isaria fumosorosea (Wize) Brown & Smith1 on Spodoptera exigua (Hübner) and Helicoverpa zea (Boddie)2

Abstract. The aim of this study was to evaluate pathogenicity and effect of four isolated native and a strain of Isaria fumosorosea on beet armyworm, Spodoptera exigua (Hübner), and corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae). Bioassays in a laboratory used neonate larvae of beet armyworm and corn earworm reared on artificial diet, which were inoculated by spraying or immersing with suspensions of 1 × 108 ml-1 conidia of I. fumosorosea, and subsequently incubated at 26°C, 65 ± 5% relative humidity, and 14:10 light: dark hours. Isolated HIB-30 killed the most immersed corn earworms, but fewest beet armyworms. HIB-30 most disrupted metamorphosis, 40%, by immersing beet armyworms, while HIB-29 and Pfr-612 least interrupted metamorphosis (8%), by spraying and immersing, respectively.

Genetic variability analysis of entomopathogenic fungi isolated from citrus-growing areas of Mexico

The objective of the study was to isolate and characterize genetically native fungi that remain in the soil of the diverse citrus regions of Mexico. We collected 142 soil samples from different Mexican states: Campeche (13), Michoacan (15), Nuevo Leon (25), San Luis Potosi (25), Sinaloa (17), Sonora (17), Tabasco (5), Tamaulipas (20), and Yucatan (5). Larvae of Galleria mellonella were used as bait to detect, trap and multiply entomopathogenic fungi in vivo. 23% of the soil samples processed were positive for the presence of entomopathogenic fungi according to the macroscopic and microscopic characteristics: Beauveria was detected in 12% (17 isolates), Metarhizium in 1% (2 isolates) and Isaria in 10% (14 isolates). We analyzed the genetic variability of the fungi by using the sequences of internal transcribed ribosomal genes (ITS-5.8S-ITS2) and subsequently performed the digestion with the enzyme Hae III. The results of the PCR generated fragments of approximately 600 bp for Beauveria spp., and Metarhizium spp. In the case of Isaria spp., it amplified a fragment above of 600 bp. Both reference strains and field isolates produced similar restriction patterns. Genera of native entomopathogenic fungi were found on the diverse citrus areas of Mexico; this opens the field for the biological control of pests affecting citrus in each region.