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Baseline susceptibility of Philippine Ostrinia furnacalis (Lepidoptera: Crambidae) populations to insecticidal Cry1A.105 and Cry2Ab2 proteins and validation of candidate diagnostic concentration for monitoring resistance

Abstract. Alcantara EP, Atienza MM, Camacho L, Parimi S. 2021. Baseline susceptibility of Philippine Ostrinia furnacalis (Lepidoptera: Crambidae) populations to insecticidal Cry1A.105 and Cry2Ab2 proteins and validation of candidate diagnostic concentration for monitoring resistance. Biodiversitas 22: 956-960. This study estimated the baseline susceptibility of Ostrinia furnacalis populations from the Philippines, to purified insecticidal Cry1A.105 and Cry2Ab2 proteins and determined a diagnostic concentration (DC) through a validation experiment. The insect populations were collected from separate sites of corn farms in Northern and Central Luzon and in South Cotabato province of the island of Mindanao. Dose-response bioassays using artificial diet surface overlay method were conducted on eight populations. The bioassay results revealed that the LC50 of Cry1A.105 and Cry2Ab2 to O. furnacalis ranged from 0.03 ng/cm2 to 0.18 ng/cm2 and 1.40 ng/cm2 to 9.98 ng/cm2, respectively. The relative susceptibility ratios between the most susceptible and most tolerant populations were 6-fold for Cry1A.105 and about 7-fold for Cry2Ab2. The candidate diagnostic concentrations (DC) based on the LC99 were calculated using the baseline bioassay data for both Cry1A.105 and Cry2Ab2. The validation was performed on populations from the same locations used in the baseline susceptibility assay and a reference strain to produce at least 99% mortality for each protein. Data showed that populations tested with Cry1A.105 produced average mortality of at least 99% for the upper limit, while this was observed in the LC99 estimate for Cry2Ab2. The validated diagnostic concentration can be used for monitoring the resistance development of O. furnacalis exposed to Bt Corn, MON89034, in the Philippines.

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Open Access
First report of Fusarium equiseti–the incitant of post flowering stalkrot of maize (Zea mays L.) in India

Maize (Zea mays L.) is one of the important cereal crops in the world and is the third largest grown cereal crop in India. Field surveys conducted in 2013-15 recorded stalk rot incidence of 28–35% in southern states of India. The typical symptoms were observed after pollination with the drying of the lower leaves and eventually entire plant wilted prematurely, lower internodes turned in to grey-green color and stalks are hollow and weak leading to the lodging of the plant. Stalk rot associated pathogen was isolated on PDA medium. Out of 219 Fusarium isolates, 19 were distinct and the fungal colonies on PDA medium showed the development of pale brown to dark brown pigment. Macro conidia were produced in orange sporodochia from monophialides on branched conidiophores with apical cells tapered and elongated. Chlamydospores were solitary and intercalary. All 19 isolates were morphologically identical, and a representative isolate was used for molecular identification. The ITS rDNA and TEF gene were amplified and sequenced using ITS1/ITS4, TEF1/TEF2primer pairs. The nBLAST search and phylogenetic analysis confirmed that the pathogen was Fusarium equiseti. Pathogenicity tests conducted on 50-day-old maize plants by injecting conidial suspension of F. equiseti produced typical stalk rot symptoms after 15 days of post-inoculation and the pathogen's identity was confirmed by cultural and morphological features after re-isolation. Association of F. equiseti as the causal agent of sheath rot of maize was reported from China. The association of F. equiseti with stalk rot of maize is the first report in India.

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A single semi‐synthetic diet with improved antimicrobial activity for mass rearing of lepidopteran insect pests of cotton and maize

AbstractHelicoverpa armigera (Hübner), Earias vittella (Fabricius), Spodoptera litura (Fabricius), Spodoptera exigua (Hübner) (all Lepidoptera: Noctuidae), Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) are the major pests of cotton and maize. Mass‐rearing of these insects under controlled conditions is necessary to obtain the numbers needed to conduct bioassays to screen insecticides, proteins, and other compounds, as tools for insect pest management. We present a diet suitable for rearing the six lepidopteran pests (five cotton and one maize pest). We further show that this diet is on par with or superior to the published diet recipes for each of the insect species, which were studied for three generations. We also discuss the advantages of antimicrobials other than formalin for keeping microbial growth under check. A combination of antimicrobial solution and benomyl provided effective control and suppressed the growth of microbes for a longer period than a formalin‐containing diet. A common diet for six pests provide opportunities for automation of diet preparation in addition to improved throughput and consistency in the process, while eliminating diet‐batch related errors.

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Open Access
Stability of expression of Cry1Ac and Cry2Ab2 proteins in Bollgard-II hybrids at different stages of crop growth in different genotypes across cropping seasons and multiple geographies.

Bollgard-II cotton expressing Cry1Ac and Cry2Ab2 insecticidal proteins has been commercially cultivated in India since 2006 to control bollworms. These genes were introgressed into parental germplasm of numerous hybrids. Therefore, it is imperative that these insecticidal proteins are expressed in sufficient quantities in different tissues, throughout the season irrespective of genetic background or environmental conditions for effective performance. Here, we document results of a comprehensive study on pattern of expression of Bt proteins across different stages of crop growth in > 2000 cotton hybrids (Gossypium hirsutum), across 12 cropping seasons tested in the Northern, Southern or Central zones in India, in terminal leaf, pre-candle square and boll epicarp tissues. Statistical analysis of variability using Linear mixed effect model was used to estimate factors contributing to variability in expression of Bt proteins. For Cry1Ac, variability was maximally contributed by genotype×season×plant growth stage effect in terminal leaves and boll epicarp, while season effect drove variability in pre-candle square. In Cry2Ab2, season effect drove variability in three tissue types. Pre-candle square tissue had most variability in expression of both proteins followed by terminal leaf and boll epicarp. Further, expression of Bt proteins in 234 G. hirsutum×G. barbadense hybrids showed similar expression patterns as intra specific hybrids though there was a significant difference in expression levels. Cry2Ab2 was expressed in significantly higher amounts when genes were in homozygous state. Bt proteins were also found to be expressed in varied amounts in different tissues and were expressed even when hybrids were grown at sub-optimal temperatures.

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