Sampling and sample collection
Metarhizium species were isolated from 3 soil types: farmland, grazing land, and forest soils. Farm soil and grazing land soil were collected from central rift valley area of Ethiopia, particularly, from East Shewa Zone located at 7.3578° N and 38.4850° E coordinates in central Oromia that the tomato is extensively produced, using an irrigation system, and T. absoluta was highly manifested without a history of biopesticide application, whereas the forest soils were collected from Menagesha National Forest with geographic coordinates of 8.9782° N and 38.5585° E that covers the altitude between 2574 to 2948 masl and located 20 km west to the capital city, Addis Ababa, Ethiopia.
Rhizospheric soil were collected from tomato farms by uprooting of aged tomato plants, whereas grazing land and forest soils were taken from undisturbed places. All types of the soil were pooled from 5 to 15 cm depth by avoiding external parts and debris in triplicate diagonals of spots by considering 5 m apart from each spot of each site. Three replicates of the soil samples from each location were composited together and only 2 kg of mixed soils composed into ethanol-sterilized (70%) plotline bag. Totally, 52 soil samples were collected from abovementioned sites and taken into Applied Microbiology Laboratory, Addis Ababa University (AAU) for further work.
Rearing of EPF susceptible insect
The great wax moth (Galleria mellonella L.) was used for EPF trapping. The insect was reared at Ambo Plant Protection Research Center (APPRC), using the methods described by Meyling (2007). Briefly, 5 pairs of female and male moths were maintained in lid-capped 500-ml conical flasks containing strip-folded tissue paper infused with honey and kept for a week to facilitate mating and egg laying. After 1 week, the folded tissue paper with lied eggs was carefully transferred into insect rearing plastic container, filled with a mix of wheat bran (100 g), honey (160 g), and glycerol (360 ml) as a food source for emerging larvae. Egg-inoculated rearing containers were incubated at 35 °C for 4 weeks until larvae were attained ages of appropriate instars of interest for EPF baiting.
Rearing of T. absoluta
The infected tomato leaves and fruits harboring larvae and pupae of T. absoluta collected from the central refit valley area using insect collection box were taken into greenhouse of AAU. The larvae and pupae were transferred into pot-grown tomato plants kept under zipped cages constructed from wooden poles and meshed cotton cloth. Pots with growing tomato plants were replaced in rearing cages once per 3 weeks for insect egg laying and for emerging larval feed. Infected tomato leaves in the rearing cages were inspected continuously for larval development and suitable larval instars were collected and used for EPF screening and pathogenicity bioassay.
EPF isolation
Metarhizium species were isolated from the soil using G. mellonella baiting method (Belay et al. 2017). Briefly, 3rd instar larvae of wax moth were shocked for 10 s in heat (65 °C) warmed water to reduce extensive waving in the soil. One kilogram of soil samples moisturized by sterilized water was filled into 1½ l capacity screw capped glass jar. Ten of heat-shocked wax moth larvae were introduced in to jars filled with soil samples separately and incubated at 28 °C for 10 days under complete dark condition. Larval death was inspected every 3 days and moisture content of the soil was adjusted by gentle moistening with sterile water each time following the inspection. The cadavers of dead larvae were carefully removed from the soil, surface sterilized by using sodium hypochlorite (3%), followed by ethanol (70%) for 10 s each, and rinsed 5 times with sterile water. Surface-sterilized larval cadavers were placed on sterile plastic plates lined with moistened tissue paper and incubated at room temperature until outgrown mycelia were sporulated under dark condition. The mycelial sporulation of EPF over the cadavers was checked, spores scraped by incinerated inoculating wire loop, transferred onto potato dextrose agar (PDA) medium, and incubated at 28 °C for 20 days. Isolates were purified by sub-culturing onto fresh PDA medium and pure cultures were maintained on agar slants at 4 °C for further work.
Morphological identification
Morphology of Metarhizium isolates were identified following the methods of Fernandes et al. (2010). The growth characteristics of isolates on the culture medium (PDA) was inspected continuously for 20 days and their colony size, mycelial color, colony reverse, and color of conidial mass were visualized. Spore feature characteristics of Metarhizium isolates particularly shape and size were examined using wet mount glass slide microscopic techniques, using light microscope (Fish Olympus phase contrast microscope).
Prescreening of isolates
Spore germination potential
Spore viability of isolates was checked through conidial germination test, using standard procedures (Habtegebriel et al. 2016). Fungal spores were collected from the 3-week-old culture by scraping with a sterilized spatula. Collected spores were added into 10 ml of sterile distilled water supplemented with Tween 80 (0.1% v/v) as a surfactant in falcon tube and evenly mixed through vortexing. Spore concentration was adjusted into 1 × 106 conidia ml−1 by using improved Neubauer hemocytometer under light microscope. A 100-μl suspension was spread over the fresh PDA and 2 sterilized glass slides were laid over inoculated medium and incubated at 25 °C for 24 h. After 24 h of incubation, over germination progresses of spores were halted by ethanol (70%) dispensing. Then 100 spores of both germinated and non-germinated were counted, following the glass slides, using × 40 magnifying objectives of light microscope and the experiment was repeated three times. Spores with germ tubes become high and then the spores itself were considered as germinated.
Sporulation rate on the agar medium
The sporulation rate of Metarhizium isolates was tested through plate culture method by incubating at 25 °C under complete dark condition. The plate cultures were checked daily for sporulation initiation since 4 days of initial inoculation. The sporulation rate of each isolate was recorded for 20 consecutive days and isolates that began to sporulate at the short time were considered as potential for pathogenicity screening procedure, using susceptible host, G. mellonella. Each plate served as a replicate and there were three replicates per isolates.
Pathogenicity screening of isolates using G. mellonella
The pathogenicity of isolates was evaluated using 3rd instar of G. mellonella larvae. Spores harvested from 3 weeks of old culture through sterilized spatula scraping were adjusted for conidial concentration at a maximum of 108 spores ml−1 as explained above and 10 ml of suspension were prepared with sterile water and Tween 80 (0.1% v/v) in sterilized falcon tube. Twenty larvae of G. mellonella were deepened into spore suspensions of each isolates for 15 s and transferred into sterile small jars filled with a mix of wheat bran (25 g), honey (40 g), and glycerol (90 ml) separately. Treated larvae-containing jars were placed at room temperature for 10 days at dark conditions. Dead larvae were collected every 3 days, surface-sterilized, and transferred into sterilized plates lined with moistened tissue paper and incubated at room temperature in complete dark condition. The moisture content of the tissue paper was adjusted using sterile water spray to enhance mycelial outgrowth over the larval cadavers. The other 20 larvae were deepened into sterilized water with Tween 80 (0.1% v/v) as control and incubated at the same condition. Treatments were repeated 3 times to minimize uncertainty (Habtegebriel et al. 2016).
Pathogenicity of isolates against T. absoluta
Pathogenicity of 13 Metarhizium isolates prescreened by the cumulative biological efficiency index model was evaluated against 2nd and 3rd larval instars of T. absoluta (Sabbour and Singer 2014). Briefly, the spore suspensions of isolates were prepared into 1 × 107 conidia ml−1 consecration, using sterile water with Tween 80 (0.1% v/v). The tomato leaves were surface-sterilized using ethanol (70%) disinfection for 3 min and rinsed 3 times with sterile distilled water. The tomato leaf petioles were tied by UV sterilized cotton wool to retain water and prevent leaf drying. The surface-sterilized leaves were maintained in sterile plastic plates and sprayed with 3 ml of concentration-adjusted fungal spores and air-dried under the safety cabinet for 3 min. Then 20 of each 2nd and 3rd larval instars of T. absoluta were released separately over the spore-sprayed leaves and incubated at room temperature for 7 days. The other 20 larvae of each instar of T. absoluta were released over surface-sterilized leaves sprayed with Tween 80 (0.1% v/v) plus water and incubated in the same condition as a control. Mortality of larvae was checked daily and dead larvae were surface-sterilized and transferred into other sterile plastic plates containing moisten tissue paper. Plates with larval cadaver were incubated at room temperature in dark conditions until developing the mycosis (Sabbour and Singer 2014). These cultures were maintained for 20 days on the plate to determine spore concentration per larval cadavers from the 3rd instar of larvae by using spore washing and microscopic counting method.
Dose response of isolates to T. absoluta
The concentrations (LC50 and LC90) of mortality response and time taken to kill 50% (LT50) for 6 of the most virulent Metarhizium isolates were evaluated (Tefera and Pringle 2004). The stock spore suspensions of each isolate were prepared in sterile distilled water containing Tween 80 (0.1% v/v). Concentrations were down-adjusted to 1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 spore ml−1 and evaluated for the 3rd instar of larvae, following the same procedure as described for pathogenicity assay. Twenty larvae were used for each concentration and control. Fungal infections of the dead larvae were confirmed after the outgrowth of mycosis on the cadavers. Mortality of larvae was recorded daily for 10 days, but lethal time calculation was performed only for three intermediate concentrations (1 × 104, 1 × 106, and 1 × 108 spore ml−1) and all treatments were replicated 3 times.
Molecular characterization of EPF isolates
DNA extraction
The genomic DNA of the fungi was extracted from 4 days old mycelial culture grown on potato dextrose agar, using a quick and safe fungal DNA extraction method (Chi et al. 2009). Approximately 400 mg of mycelia grown on the PDA was transferred into 1.5 ml Eppendorf tube containing 0.5 ml of DNA extraction buffer (1 M KCl; 100 mM Tris-HCl; 10 mM EDTA) using a sterile toothpick. Soon after mycelia transfer, mycelial tissue was pulverized by using sterile plastic pestle fitted with an electronic instrument Black and Decker portable electronic drill (American manufacturer of power tools, Stanley Black & Decker, Inc.) for 2 to 3 s. Mycelial lysates were centrifuged at 12,000g for 10 min in order to separate cell debris and contaminants from the supernatant. The DNA containing supernatant was carefully transferred into another 1.5 ml Eeppendorf tubes containing 0.3 ml of 2-propanol and mixed through tube inverting and centrifuged at 13,000g for 10 min. After discarding the supernatant, pellet in the Eppendorf tube was gently washed with 0.7 ml of ethanol (70%) and allowed for ethanol evaporation at room temperature for 15 min. Then DNA pellet was dissolved by using 100 μl of 20 mM Tris solution through gentle tapping and stored at – 20 °C for PCR use. The DNA amount in the suspension was quantified by transferring 2 μl of aliquots on to nano-drop microplates in duplicate using an instrument, BioTek Synergy2TM Multi-mode Microplate Reader controlled by Gen5TM Dada analysis software, USA. Furthermore, DNA purity was confirmed by running PCR products under 1% agarose gel electrophoresis.
PCR amplification
The PCR amplification of DNA was performed by using ITS1 and ITS4 primers. Primers ITS1 TCCGTAGGTGAACCTGCGG forward and ITS4 TCCTCCGCTTATTGATATGC reverse were used to amplify the target regions (White et al. 1990). The PCR amplification reaction was conducted in a total volume of 20 μl. The master mix was prepared from the components of 6.8 μl of water, 4 μl of buffer, 1 μl (2.5 mM μl−1) MgCl2, 1 μl (0.5 mM μl−1) of dNTP, 0.2 μl (1 U μl−1) of GoTaq polymerase, 2.5 μl (2.5 μM μl−1) of each ITS1 (forward) and ITS4 (reverse) primers, and 1 μl (30 μg μl−1) of genomic DNA. PCR thermocycler settings were 4 min initial denaturation at 94 °C followed by 35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 56 °C, and 1 min extension at 72 °C. The final extension was performed at 72 °C for 5 min with a storage temperature of 4 °C. The PCR products were assayed by electrophoresis on a 2.5% agarose gel with TBE buffer (Tris; Borate; EDTA) at 100 V for 55 min. Then the gel was stained by shaking within 200 ml of TBE buffer supplemented with 10 μl (v/v) of non-carcinogenic dye “SafeViewTM Plus” for 50 min and photographed under UV light using eight-mega pixels canon pc1201 digital camera. The PCR amplicons amplified in a volume of 50 μl was purified by using NucleoSpin® Gel and PCR Clean-upkits (Germany), checked for DNA purity using 2.5% agarose gel electrophoresis, and send to Macrogen Inc. Seoul, Korea for sequencing.
Data analysis
The obtained data for larval mortality were calculated by using Abbott’s formula (Abbott 1925). Spore germination, sporulation date, and screening test results were analyzed by using one-way of variance (ANOVA) using SPSS software version 25 statistical programs. Mean separations were calculated using Tukey’s HSD test when the value were significant at 푝 = 0.05. The relative sporulation rate (RSR) of the isolates was calculated using the formula \( \mathrm{RSR}=\frac{\mathrm{Exprimental}\ \mathrm{lasting}\ \mathrm{date}}{\mathrm{PCS}\ \mathrm{date}\ \mathrm{of}\ \mathrm{isolate}\ } \) where PCS, plate culture sporulation, and biological efficacy (BE) index were computed by using the biological indexing formula of Sain et al. (2019) with some modification: BE index (%) = 37 (SG) + 13 (RSR) + 50 ( LM) where SG, spore germination and LM, larval mortality in 7 days post inoculation of spores. Lethal concentration (LC50 and LC90) was analyzed by using Probit analysis software in SPSS version 25. The dose responses of each replicate were checked for estimation of lethal time to kill 50% (LT50) of exposed larvae. Analysis of variances and means were separated by using least significant differences (LSD).