Rearing of the spiny
Full-grown SBW larvae of field strain were collected from infested cotton bolls in Sharkia Province, Egypt, and reared in the laboratory at the Bollworms Research Department, Plant Protection Research Institute, Agriculture Research Center, Giza, Egypt, for 6 generations. The neonate larvae were transferred into glass tubes (2.5 × 7cm) containing about 4 g semi artificial diet Shorey and Hale (1965). The experiments were performed at constant temperature of 26 ± 1 °C and 75 ± 5% RH. The diet for maintaining laboratory colony preparing by adding boiled water to 250 g kidney beans and 125 g wheat grated then add over heat for 70 min, lifted and left for 20 min to be cooled and clarifying water from them. The diet was blended by 100 ml milk in an electric blender and placed in the refrigerator for 24 h. After that 49 g dry active yeast, 3 g ascorbic acid, 1.75 g sorbic acid, 1.75 g methyl parahydroxy benzoate, 8 ml mixture of vitamins, and 2.5 ml formaldehyde 34–38% were added, all thoroughly blended and kept in the refrigerator for 24 h before being used (Amer and El-Sayed 2015).
The culture was away from any contamination with any microorganisms or pesticides. The dead larvae in culture were obtained at full grown and stored slowly in sterilized tightly closed vials at 4 °C in a refrigerator until needed (Mahfouz and Abou El-Ela 2011).
Microbiological analysis
Fungi isolation technique
In order to reveal any microorganisms associated with the dead spiny bollworm SBW larvae (4th instar), each of the refrigerated individuals was examined through 24–72 h from the time of storage under aseptic conditions. The larvae were surface sterilized by dipping in 2% sodium hypochlorite for 3–5 min to isolate fungi on insect’s surface, then passed through 5 separated washings with sterile distilled water (Crecchio and Stotzky 2001). For insuring the appropriate surface sterilization, checks were made by spreading the last washing solution on Czapek- Dox agar medium. Sterilized larvae were dried up between 2 filter papers (Whattman No. 1), then transferred aseptically into a sterile mortar and macerated with a sterile pestle, diluted and plated on Czapek- Dox agar medium for growth incubating at 30 °C for 5–7 days. Incubated plates were inspected daily to observe the colonies growth that were then purified and stored on slants of the desired artificial media at 4 °C. The isolates were cultured periodically until they had been used in the subsequent experiments. Healthy larvae were subjected to the same procedures of isolation for obtaining the expected dormant pathogens.
Screening of fungal isolates for their mortality effect on E. insulana
Spore suspensions were obtained by washing the 7-day-old slant of tested fungal isolates (Dulmage et al. 1971; Mohd-Salleh and Lewis 1983), then inoculated a 100 ml of Czapek- Dox agar medium (Oxoid 1982) composed of (g/l) 20 sucrose, 2.0 NaNO3, 1.0 KH2PO4, 0.5 MgSO4·7H2O, 0.5 KCl, and 20.0 agar-agar and dissolved in 1 l tap water, pH 5.0 in a 250-ml Erlenmeyer flask with each suspension. The inoculated broth was incubated at 30 °C for 7 days, while metabolites were obtained by filtration using the filter paper (Whattman No.1.). Spore suspension and filtrate of all isolates were tested for their mortality effect and on biological aspects of E. insulana as described in the bioassay method.
Bioassay
Two milliliters from each spore suspension and metabolites was mixed with the artificial diet in each dish, while the diet of control was mixed with water only. Each treatment was replicated 3 times. Batches of 20 1st instar larvae were transferred immediately after hatching using a fine brush to each treated Petri dish after about 30 min from mixing in the diet. Treated Petri dishes were covered by a fine and soft paper below the glass cover to prevent larvae to escape. All treatments were incubated at the constant conditions of 26 ± 1 °C and 70 ± 5% RH. After 24 h of exposure and feeding, dead and alive larvae were counted. The mortality percentages were calculated.
$$ Larval\ mortality\%= dead\ larvae/ total\ larvae\times 100 $$
Mortality data were corrected according to Abbott (1925).
$$ Corrected\ mortality\%=\frac{mortality\ in\ treated- mortality\ in\ control}{mortality\ in\ control}\times 100 $$
The remained alive larvae of each treatment were transferred singly to glass tubes (2 × 7.5 cm) containing about 4 g of untreated control diet and covered with a piece of absorbent cotton and held under the same conditions as mentioned above. Larvae were examined daily to record the biological parameter, larval duration and pupation percentage; then, pupae were transferred individually to other clean tubes and incubated until moth emergence. Pupal duration, adult emergence percentage, sex ratio (as females), and deformed adults were calculated. Emerged moths from each treatment were sexed and caged in 2 pairs, and eggs deposited on strips of muslin cloth hanged in the chimney cages.
Forty pairs were used from each treatment (male and female) under the previously mentioned rearing conditions. A piece of cotton wool previously soaked in 10% sugar solution was hung inside the jars near its upper opening for moth feeding and changed by new one every 2 days. The upper openings of cages were covered by muslin cloth followed by a tightly secured paper with rubber bands. Each cage was examined daily to record data of several biological aspects such as preovipositional, ovipositional periods, number of deposited eggs, postovipositional period, and longevity of males and females. The deposited eggs were collected daily from strips of muslin cloth then transferred to a convenient glass jar and incubated at the same conditions to record hatchability percentages.
Characterization of most potent fungal isolate
Identification of isolated fungi by light microscope
The developed fungal colonies were examined daily, and the purified fungi were identified to the species level whenever possible. The identification of fungal genera and species was carried out by the help of the following universally accepted keys for identification of the different isolates. Morphology based on colony shape, height, and color of the aerial hyphae as well as the base color, growth rate, margin characteristics, surface texture, and depth of growth into the medium. Tests were contrasted with an ordered key for the genus Acremonium sp. (Rifai 1969).
Molecular characterization (sequence of 18S rRNA gene of DNA)
Sequence of 18S rRNA gene of DNA of fungal isolates was done at Sigma Scientific Services Co, Cairo, Egypt, also kindly confirmed by Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt (Figs. 1 and 2). Molecular characterization involved the following steps according to the protocol adopted by Woese and Fox (1977) and Abdel-Salam (2003).
Screening of lipase, protease and chitinase produced by Acremonium sp.
In vitro
Seven-day-old fungal culture was used as a standard inoculant. At the end of incubation period for each enzyme (protease, lipase and chitinase) respectively, the fungal cultures were filtered and the clear supernatants were considered the source of crude enzyme (Reda et al. 2013).
The most active isolate of Acremonium sp. was screened for lipase, protease, and chitinase production according to clearing zone technique using Dox-yeast extract-tributyrin agar (Elwan et al. 1977); Dox agar with replacing of NaNO3 by 0.2% gelatin (Ammar et al. 1991) and chitin media which consists of (g/l): colloidal chitin, 0.5; yeast extract, 0.5; (NH4)2SO4, 1.0; MgSO4. 7H2O, 0.3; K2HPO4, 1.36; agar-agar, 20 (CM) (Rajamanickam et al. 2012), respectively.
In vivo
The culture filtrates of lipase, protease, and chitinase media of tested strains after incubation for 7 days at 30 °C were obtained and screening against SBW.
Statistical analysis
Obtained results were analyzed according to Little and Hills (1975), using CoStat computer program Cohort Software, P. O. Box 1149, Berkeley CA 9471 (CoStat Statistical Software, 2005).
