Sources of insects and fungi
Cultures of M. persicae and A. fabae were established with individuals collected initially from greenhouses at the Faculty of Agriculture, University of Kufa, Iraq, in 2018. The aphids were identified under a dissecting microscope, using the keys by Blackman and Eastop [14]. Cucumber, Cucumis sativus L. cultivar “Babylon” (Peto Seeds Company, Inc., Chicago, IL), were grown in the growth chamber for maintaining the aphid cultures and for the experiments. The aphid cultures were maintained in 45 × 45 × 45 cm cages at 23 ± 2 °C and 16:8 (L:D) h photoperiod for several generations. Plants were replaced every 2 weeks with uninfested 3–4-week-old plants.
Cultures of C. rosea were established by culturing dead M. persicae adults collected from greenhouses at the Faculty of Agriculture, University of Kufa (25 cadavers), and the Faculty of Agriculture, University of Basra, Iraq (25 cadavers). The aphid cadavers were surface sterilized with 1% sodium hypochlorite for 1 min, followed by washing with sterilized water and transferred to SDAY media. Plates, each with 10 cadavers, were incubated at 25 ± 1 °C for 7 day. A single tip from the mycelium were cut and sub-cultured on new plates for purification, and then stored at 4 °C as pure cultures. Morphological identification of fungal isolates were performed using Schroers [15] method. Aerial conidia were harvested from 10-day-old cultures by adding 12 ml 0.01% v/v Tween 80 (BDH Chemicals Ltd., Poole, UK) into the culture agar plates, then gently scraping the surface of the cultures with a sterile inoculating loop to dislodge the conidia from the surface. The conidial suspension was pipetted from the plate and filtered through three layers of sterile cheesecloth. The number of conidia in the suspension was determined using a hemocytometer (Neubauer Improved; Superior Marienfeld, Königshofen, Germany). The resulting suspension was diluted to the desired concentrations.
Molecular identification and phylogenetic analysis
Genomic DNA of representative strains of C. rosea was extracted from freshly collected mycelium of 7-day-old cultures using a DNA extraction kit (Favorgen; Biotech Corp., Pingtung, Taiwan) following the manufacturer’s instructions. A spectrophotometer (NanoDrop 2000; Thermo Scientific, Waltham, Massachusetts) was used to quantify the extracted DNA and equalled with distilled water. The internal transcribed spacer (ITS) region of fungal isolates was amplified using polymerase chain reaction (PCR) with primer pairs ITS1 (TCCGTAGGTGAACCTGCGG and ITS4 (TCCTCCGCTTATTGATATGC) [16]. PCR was carried out in 20 μl reaction mix containing 1 μl ITS1 primer, 1 μl ITS4 primer, 12 μl GREENTaq DNA polymerase, 4 μl ddH2O, and 2 μl of template. PCR was performed with initial denaturation for 5 min at 98 °C, followed by 36 cycles of denaturation for 40 s at 94 °C, primer annealing for 40 s at 55 °C, and extension for 1 min at 72 °C. A final extension for 5 min at 72 °C was performed. The PCR products were run in electrophoresis-agarose gel at 1.5%, stained with 0.4 μg/ml ethidium bromide, and bands visualized with a UV illuminator (ULTRA TEC Manufacturing, Santa Ana, CA). PCR product was cleaned up with Exosape (Applied Biosystems, Foster, California) and sequenced by Macrogen Company (Seoul, South Korea).
Referenced sequences of similar regions from the genus Clonostachys were obtained from National Center for Biotechnology Information (NCBI) database [17]. Similar sequences were compared with sequences placed in GenBank databases by using of the Basic Local Alignment Search Tool (BLAST) [17]. Related sequences were aligned using DNA Baser software and a phylogenetic tree was built with Geneious software version 10.2.3 [18].
Virulence bioassays
This experiment was conducted to evaluate the virulence of AA80 and AA82 isolates against adults of M. persicae and A. fabae. In order to obtain a uniform age (1–2-day-old) of adult M. persicae and A. fabae for use in all experiments, 20 adults of each species were transferred from the stock cultures onto each 4-week-old cucumber plants, isolated in 3-cm-diameter clip-cages, and allowed to produce nymphs for 1 day. After the removal of the adults and excess nymphs, 25 nymphs were left on each plant and allowed to develop for nine additional days before treatment in a growth chamber (Binder Ltd., Suffolk, UK) at 20 ± 2 °C, 75 ± 2% RH and a photoperiod of 16:8 h (L:D). The clip cages were removed before the treatment.
Two milliliters of conidia suspensions (1 × 105, 1 × 106, 1 × 107, 1 × 108, or 1 × 109 conidia ml−1) of either C. rosea isolate AA82 or isolate AA80 were applied onto each infested 4-week-old potted cucumber plant using a hand-held sprayer (Ampulla, Hyde, UK). Plants assigned to the control treatment were sprayed with 0.02% aqueous Tween 80 solution. Eight replicates were carried out for the control and each concentration of each fungal isolate against either M. persicae or A. fabae. Plants were air-dried on bench top at room temperature for 1 h. The treated plants were then transferred to the growth chamber maintained at 26 ± 1 °C, ≥ 70% RH and a photoperiod of 16:8 (L:D) h. To prevent aphids from moving between treated plants, the aphids were kept in the 3-cm-diameter clip-cages post-spraying. Aphid mortality was recorded at 1, 3, 5, 7, and 10 day after application. Dead aphids were surface sterilized by rinsing twice with 70% ethanol for 30 s and then with sterilized distilled water, after which they were placed on water agar (3 g of agar per L of water) in Petri dishes for 5 day to confirm infection by C. rosea [19]. A cadaver was regarded as dead from infection by these fungi if the conidia were recovered from the cadaver. Median lethal concentration (LC50) and median lethal time (LT50) were calculated.
Effect of fungal infection on aphid fecundity
This experiment was conducted to determine the effect C. rosea infection on nymph production by M. persicae and A. fabae adults. The method described above was used to obtain uniform age of adults of either M. persicae or A. fabae. The LC50 value of each isolate was sprayed once onto infested leaves of cucumber plants (2 ml per leaf) each harboring 20 adult M. persicae or A. fabae. Plants assigned to control treatment were sprayed with 0.02% Tween 80 solution. After treatment, the plants were kept in the laboratory at room temperature for 1 h to dry, and then transferred to a growth chamber maintained at the same environmental conditions described above. After 1 day, a treated aphid (5 aphids from each plant leaf) was selected randomly and transferred with a fine camel hairbrush onto an uninfested cucumber plant and isolated with a new 3-cm-diameter clip-cage (2 clip-cages per plant). A total of 25 individuals of each aphid species were isolated in this manner for each fungal isolate treatment. Plants were then transferred to the same growth chamber maintained at the same conditions described above. Each leaf clip-cage was carefully opened and inspected daily until the death of the aphids and the number of nymphs produced by each individual was recorded.
Statistical analysis
Statistical analysis was performed using GenStat software (VSN International 2016). Cumulative mortality was corrected for control mortality by removing the number of dead aphids in the control data from the corresponding number. Normal distribution of data was assessed using the Shapiro-Wilk test. Mortalities were logit transformed when necessary to meet the assumption of normality. LC50, LT50, and 95% confidence limit of each C. rosea isolate against each aphid species were calculated by probit regression analysis. Two-factor repeated measure ANOVA was used to determine the effect of fungal isolate and aphid species on aphid mortality. The effect of fungal infection on aphid fecundity was analyzed separately for each aphid species using one-way measure ANOVA. Mean comparisons were performed using Turkey’s test at P = 0.05.