Effects of Myco-Entomopathogens on Bio-Control of Trialeurodes Vaporariorum, Endophytic Colonization of Tomato, and Compatibility with selected pesticides

doi:10.21203/rs.3.rs-1835624/v1

Purpose

The biological pest control efficiency of entomopathogenic fungi is improved by the endophytic establishment and combination with agricultural recommended pesticides and plant extracts. This study aimed to evaluate the effects of B. bassiana and M. anisopliae on biological control of T. vaporariorum, endophytic colonization, and compatibility with selected pesticides.

Methods

The endophytic colonization ability of entomopathogenic fungal isolates was evaluated by using soil drenching and foliar spraying methods under greenhouse condition. Compatibility interaction of potential entomopathogenic fungi with selected pesticides was calculated based on the biological index formula.

Results

The endophytic colonization pattern of B. bassiana and M. anisopliae isolates using soil drench and foliar spray inoculation methods exhibited significant differences in the different parts of tomato plants. The endophytic B. bassiana and M. anisopliae isolates induced a significant reduction in the number of nymphs and adults of T. vaporariorum (p < 0.001). Thus, the application of B. bassiana AAUMB-29 reduced nymphs by 67% while M. anisopliae AAUDM-43 reduced adults with a percentage reduction of 52% over control. The compatibility study revealed that all tested concentrations of neem extract and chemical pesticides were caused by different levels of inhibition on vegetative growth, germination, and sporulation of M. anisopliae AAUDM-43, B. bassiana AAUMFB-77, and B.bassiana AAUMB-29. The biological index vales indicated that all isolates were compatible with neem oil, neem seed crud extract, and chemical insecticides at both half and recommended doses.

Conclusion

The tomato endophytic B. bassiana AAUMB-29 and M. anisopliae AAUDM-43 were convinced significant reduction of T. vaporariorum, and highly compatible with selected pesticides. These potential isolates should be used in integrated pest management programs

Biological index

Endophytic colonization

compatibility

B.bassiana

M. anisopliae

Entomopathogenic fungi (EPF) are cosmopolitan and natural enemies of wide ranges of agricultural pests that infect insects by penetrating their cuticle to enter their bodies to eventually kill them and feeding on them (Mora et al., 2017). According to (Maina et al., 2018) about 800 fungi species are identified as pathogenic for insects from which many commercial mycoinsecticides are being developed from three genera/species of entomopathogenic fungi Beauveria bassiana, Metharhizum anisopliae, and Isaria fumosoroseus. These fungi produce conidia that attach and penetrate insect cuticles and through enzymatic production invade and kill the insects (Brunner-Mendoza et al., 2019).

Apart from suppressing insect activities, entomopathogens are known to be endophytes to promote plant growth by having different plant growth-promoting properties (Bamisile et al., 2018). The endophytic entomopathogenic B. bassiana and M. anisopliae and are conduits to provide insect-derived nitrogen to plant hosts (Behie et al., 2015). In general, entomopathogenic fungi as endophytes stand out as a prospective approach for plant protection, inducing resistance against insect pests and consequently reducing pesticide use (Vidal and Jaber, 2015).

Whiteflies are soft-bodied, winged insects closely related to aphids and mealybugs and known to severely affect tomatoes, eggplant, peppers, okra, and sweet potatoes (Jha and Kumar, 2017; Zafar et al., 2016a). They contribute to severe damage and yield losses of plants due to sucking juices from them, causing leaves to yellow, shrivel, and drop prematurely, and transmission of most viruses such as Crinivirus and Torradovirus onto tomato and other plants (Navas-Castillo et al., 2011). The Trialeurodes vaporariorum is one of the whitefly species that damage tomato due to its prolificacy and yield losses in both greenhouse and field production.

Tomato plant tissues have been naturally and artificially colonized with entomopathogenic fungi and they can promote growth and protect plants from insect pests (Silva et al., 2020). According to (Barra-Bucarei et al., 2020), the endophytic B. bassiana has the potential to control T. vaporariorum and promote the growth of tomatoes. (Kabaluk et al., 2007) also showed that M. anisopliae is an important endophyte that promotes plant development, increases the seed germination rate of tomato, and protect corn plant against coleopteran pests. In addition to endophytic development, the combined use of endophytic entomopathogenic fungi with other compatible products might produce higher control efficacy levels on tomatoes. It is established that biological control of whiteflies is a sustainable pest management option for increasing vegetable production. Using entomopathogens as bio-pesticides in pest management is called microbial control, which can be a critical part of integrated pest management (IPM) against several pests(Ali et al., 2018).

The bio-control efficacy of entomopathogenic fungi combined with agricultural recommended pesticides (Yii et al., 2015) and plant extracts such as neem (Shoukat et al., 2016) are found to be an effective strategy for the control of pests and diseases (Jaber and Ownley, 2018). Thus, the comprehensive study of the compatibility of different treatments of entomopathogenic fungi with chemical insecticides and neem-based insecticides helps to choose the optimal combination to improve integrated pest management efficiency. Abidin et al. (2017) reported that the compatibility of chemical pesticides and entomopathogenic fungi, B. bassiana and M. anisopliae exhibited variation in the percentage of germination, percentage of inhibition, and sporulation. Although the application of M. anisopliae and B. bassiana as biocontrol agents is known for many years direct application of these fungal entomopathogens is affected by UV radiation, relative humidity, and temperature that compromise their bio-control effectiveness (Kim et al., 2013; Wu et al., 2020).

As a result, there is a need for research for identifying entomopathogens and their compatible IPM components to test their efficacy under the objective agro-ecological conditions concerning their endophytic establishment and application as effective biological pest control agents. The objective of this study is, therefore, intended to evaluate the effects of B. bassiana and M. anisopliae isolates on the biological control of T. vaporariorum, endophytic colonization of tomato, and compatibility interaction with Neem extracts and selected chemical insecticides.

Isolation and identification of entomopathogenic fungal isolates

Indigenous entomopathogenic fungal isolates were detected from soil samples collected from tomato-growing farmlands and forest sites of Ethiopia using the Galleria mel1onella baiting method (Meyling and Eilenberg, 2006). The genomic DNA was extracted from entomopathogenic fungal isolates following the protocol of (Chi et al., 2009). The internal transcribed spacer (ITS) regions of rDNA of the fungal isolates were amplified and sequenced as described by (White et al., 1990). All sequences were compared with already published sequences of known fungi from the NCBI GenBank Database. Phylogenetic and molecular evolutionary analysis was constructed by using Molecular Evolutionary Genetics Analysis (MEGA-X-10.1.8) software (Tamura et al., 2007). The spore suspension was prepared following the protocol of (Lacey, 2012). Fungal conidia were harvested by gently scraping the surface of 14-day old sporulating cultures of B. bassiana and M. anisopliae after flooding the plates with sterile distilled water containing Tween 80 (0.1% v/v). The resulting mycelia and spores were then filtered through several layers of sterile cheesecloth to remove any mycelial fragments. The conidial suspension of each isolate was homogenized with a magnetic stirrer for 5 min. The suspension concentration was determined under the microscope using a Neubauer hemocytometer and adjusted to a concentration of 1×10⁷ conidia ml⁻¹. A total of five entomopathogenic fungal isolates were used in the study (Table 1).

Test whitefly species

The Whitefly adults (B.tabaci, and T.vaporariorum) were collected from Meki during 2018 from a tomato field using a manual aspirator and identified based on their morphological basis (Martin et al., 2000). They were primarily detected with hand lenses (20X) in the field followed by laboratory identification using a stereomicroscope.

Sources of chemical pesticides

The two chemical pesticides of Hunter 40 EC and Ethiothoate 40% EC were selected for they are commonly used for the management of whiteflies in the Rift Valley of Ethiopia. These chemical pesticides were purchased from the local market and used to check their compatibility test at three different concentrations, recommended dose, half of the recommended dose, and double of the recommended dose.

Preparation of plant material

Undamaged ripe seeds of Neem (Azadirachta indica) were gathered from neem trees from Raya Kobo, North Wollo, Amhara region, Ethiopia. The seeds were washed thoroughly three times with water to remove surface dirt and impurities. They were air-dried to a constant weight under shade for 10-15 days separately (Vasantharaj et al., 2013). After drying, the plant materials were powdered by an electric grinder and sieved using sieves (500μm opening) to remove coarse particles. The samples were sealed in polythene bags until the time of extraction.

Preparation of neem seed crude extracts

Crude plant fractions were extracted using the modified method by (Chonoko and Rufai, 2011). The neem seed powder was then mixed with ethanol in Erlenmeyer flasks with a 1:10 solute to solvent ratio and kept in a shaker (GFL, model 3020, Germany) under 120 rotations per minute at room temperature for 72 hours. The macerated samples were first filtered through a four-fold muslin cloth and the supernatant was filtered using a filter paper (Whatman No.1Whatman Ltd., England) and concentrated in a vacuum at 45°C using a rotary evaporator (a brand of BUCHI Germany) at 60 rotations per minute. The crude extracts were further allowed to fully dry with oven at 45^oC and the extracts were stored at -20ºC for future tests.

Extraction of neem seed oil

The seed oil was extracted from neem seed powder by using the soxhlet apparatus at room temperature as described by (Kebede et al., 2010). Thus, 100g of seed powder was extracted with 1000ml of Ethanol + n-Hexane (50:50%), from which the solvents were removed by rotary evaporation at a temperature below 45^◦C. Finally, neem oil adjusted at recommended dose (RD), half recommended dose (1/2RD), and double recommended dose (2RD) for compatibility test (Table 1).

Preparation of entomopathogen inoculums

The spore suspension of each entomopathogen was prepared following the protocol of (Lacey, 2012). Conidia were harvested by gently scraping the surface of 14-day old sporulating cultures of each fungus after flooding the plates with sterile distilled water containing Tween 80 (0.1% v/v). The suspensions were then filtered through several layers of sterile cheesecloth to remove any mycelia fragments. Each conidial suspension was homogenized with a magnetic stirrer for 5 min, and the concentration was adjusted to 1×10⁷ conidia ml⁻¹ under the microscope using a Neubauer hemocytometer.

Table 1: Entomopathogenic fungal isolates, botanical and chemical pesticide used

Fungal isolate	Species			Accession number		Collection sites
AAUMB-21	B. bassiana			MW077112		East Shoa, Ethiopia
AAUMB-29	B. bassiana			MW077113		East Shoa, Ethiopia
AAUMFB-77	B. bassiana			MW077115		West Show, Ethiopia
AAUDM-43	M. anisopliae			MW077119		East Shoa, Ethiopia
AAUMFM-6	M. anisopliae			MW077120		WestShow, Ethiopia
Botanical insecticides		Concentration (%)
		Control		1/2RD	RD		2RD
Neem seed oil		0		0.5	1		2
Neem seed crude extract		0		2.5	5		10
Chemical insecticides	Application dose
	control		1/2RD		RD			2RD
Hunter 40 EC	0		1.25ml/L		2.5ml/L			5ml/L
Ethiothoate 40% EC	0		2.5ml/L		5ml/L			10ml/L

RD=recommended dose

Endophytic colonization of entomopathogenic fungi on tomato

Tomato seed preparation

Tomato seeds of melkashola variety were surface-sterilized according to (Parsa et al., 2016). They were surface sterilized using 0.5 % sodium hypochlorite (NaOCl) for 3 min and 70% ethanol for 2 min each. The seeds were rinsed 3 times with sterilized distilled water and dried on sterile absorbent paper for 1 h. Finally, ten µl of water were taken from the rinsed and cultured in Petri dishes with PDA containing chloramphenicol to verify the quality of the disinfection process.

Inoculation methods

The surface-sterilized tomato seeds were sown into 330cm³ plastic pots in triplicates containing a sterilized mixture of garden soil, compost, and vermin-culture in the ratio of 1:1:1. All fungal isolates were inoculated with soil drenching and foliar spraying after 21 days after planting of tomatoes under greenhouse conditions (Afandhi et al., 2019). The soil drench inoculation method was performed by inoculating the roots of each seedling with 10 ml conidial suspension (1×10⁷ conidia ml^-1) of isolates while the 10ml of sterile distilled water was applied for control. . For the foliar spray inoculation method, 10ml conidial suspension (1×10⁷ conidia ml^-1) was applied directly to leaves of seedling with a hand sprayer. All pots were covered with aluminum foil during inoculation to prevent conidial suspension runoff to the soil and maintained under greenhouse conditions for four weeks.

Assessment of endophytic colonization

After four weeks of inoculation, tomato leaves, stems, and roots were evaluated for endophytic colonization of entomopathogenic fungi in the tomato plant according to (Posada et al., 2007). The plant parts were washed with tap water and cut into 20mm pieces by using a sterile scalpel. They were surface-sterilized with 0.5% NaOCl for three minutes, 70% ethanol for two minutes, and rinsed three times with sterile distilled water. Each portion of the leaves stems and roots were further cut into small pieces (5mm pieces) from which, 6pieces were transferred into PDA and incubated at 25^oC under complete darkness for three weeks. Colonization was assessed based on the mycelial growth of the fungal inoculants, and colonization rates of entomopathogenic fungi on tomato plants were calculated (Petrini and Fisher, 1986) by using the following formula:

The effects of entomopathogenic fungi on pathogenicity of Trialeurodes vaporariorum

The adult whiteflies were mass-reared in whitefly-proof cages on potted young Melkashola tomato variety under greenhouse conditions (Zafar et al., 2016b). Tomato seedlings were foliar sprayed with an average of 10ml of 1×10⁷ conidia ml^-1ofeach strain from B.bassiana and M.anisopliae. The control seedlings were sprayed with sterile distilled water containing 0.1% Tween 80 solution. The treated plants were placed in whitefly-proof cages and, 10 newly emerged adults were introduced per seedlings, and the cages were kept under a greenhouse for 30 days. The effects of endophytic strains of the entomopathogens on the pathogenicity of whitefly T.vaporariorum was determined by counting the number of nymphs and adults of whitefly per 3 leaves in each plant (Zhang et al., 2018).

Effect of selected pesticide and neem products on conidial production, germination, and vegetative growth of entomopathogenic fungi

The effect of pesticides and neem products (compatibility) on spore germination of the entomopathogenic fungi was tested following the protocol of (Yii et al., 2015). One ml conidial suspension (10⁷ conidia ml^-1) each from B. basiana and M. anisopliae was mixed with an appropriate dose of pesticide and neem extracts in 20 ml of warm (45°C) PDA plates. The PDA medium without insecticides was inoculated with the fungal conidia suspension as the control and all treatments and control were incubated at 25°C for 24 hours. Then, 1 cm²piece block from each plate was cut out and stained with lactophenol cotton to assess spore germination. Each treatment was replicated three times. The percentage of spore germination was counted based on the number of conidia producing germ tubes per 100 counted under light microscopes (400×).

The Percentage germination was calculated using the following formula:

PG=percentage of germination, GS=Germinated spore, and NGS=non germinated spore

The effect of chemical pesticides and neem extracts on the vegetative growth and sporulation potential was determined using the poisoned food technique (Moorhouse et al., 1992). Thus, Isolates were cultured on PDA at 25^oC for 20 days. The PDA media were autoclaved and cooled to 45^oC. The pesticides and the neem extracts with appropriate concentrations (Table 6.1) were added to approximately 20 mL of each on warm PDA media and poured into the Petri dish. The same amount of medium without the pesticide was used as a control. Then 5-mm diameter agar culture blocks were transferred to the standard insecticide-PDA and Neem-extract containing media. Non-insecticide PDA and non-neem extract media were also included as a control. The plates were sealed with parafilm and incubated at room temperature for 14 days. This treatment was replicated three times.

The effect of pesticides on sporulation (compatibility) was determined from 15 days old culture as described by (Mustafa and Kaur, 2009). To that end, 5mm agar discs were randomly taken with the help of a cork borer and placed in 10ml of 0.01% Tween 80 solution and vortexed for five minutes to suspend the spores. The number of spores was determined under the light microscope at a 400× magnification by using a Neubauer hemocytometer.

Biological index for compatibility of pesticide

Compatibility interaction between entomopathogenic fungi with chemical pesticides and neem extract was calculated based on the biological index formula proposed by (Alves et al., 2007). The compatibility of biopesticides and chemical pesticides were calculated with the following formula (Alves et al., 2007):

Where; BI= biological index, VG=vegetative growth; GR=conidia germination; SPR=sporulation. The biological index classification was categorized as toxic (BI < 42), moderately toxic (42 ≤ BI ≤ 60), and compatible (BI>60). The average percentage of vegetative growth, sporulation, and germination treatments were corrected to the respective controls before calculating the biological index.

Data analysis

All data collected were subjected to normality and homogeneity test of variances using Q-Q plot, Levene’s homogeneity test, and Shapiro–Wilk Normality test before any data analysis. The mean values were compared by using the Tukey multiple range tests (P ≤ 0.05) using SPSS version 20.

Molecular identification

Entomopathogenic fungal isolates were genetically identified by using rDNA-ITS regions. The PCR amplification of the ITS region of rDNA give rise to a single product that produced approximately 545bp fragment size for all isolates and phylogenetic analysis confirmed that isolates were identified as Beauveria bassiana (Figure 1) and Metarhizium anisopliae (Figure 2).

Endophytic colonization

The ability of the entomopathogenic fungi, B. bassiana, and M.anisopliae strains to colonize tomato plants was assessed following foliar spray and soil drenching inoculation methods (Table 2). The success of B. bassiana (Figure 3) and M.anisopliae (Figure 4) strains colonization was confirmed by re-isolation of inoculated fungal mycelia from the surface of sterilized plant parts (leaf, stem, and root) plated on potato dextrose agar (Figure 3 and 4).The colonization pattern of the different isolates showed a significant difference in the different parts of the plants and the two methods used in the application (treatments). Under the circumstances, the isolates showed better colonization through the foliar spray method than they did through soil drenching; where two isolates, B. bassiana AAUMB-21 and M. anisopliae AAUMFM-6 failed to colonize the stem and root parts. After four weeks of foliar spray, B. bassiana AAUMB-29 (89.28%) B. bassiana AAUMFB-77(53.57%), and B. bassiana AAUMB-29 (78.57%) resulted in better colonization of root, stem, and leaf respectively. Under the soil drenching method, B. bassiana AAUMFB-77 (49.99%) achieved better root colonization, and B. bassiana AAUMB-29 induced the highest colonization of stem (32.14%) and leaf (17.86%) on tomato seedlings, respectively.

Table 2: Colonization percentage of B. bassiana and M.anisopliae strains on root, stem, and leaf of tomato seedlings

Isolates	Colonization frequencies (%)
	Foliar spray			Soil drenching
	Root	Stem	Leaf	Root	Stem	Leaf
B. bassiana AAUMB-29	44.62±6.12^a	78.57±4.12^a	89.28±3.57^a	39.28±8.99^a	32.14±6.84^a	17.86±6.83^a
B. bassiana AAUMFB-77	53.57±6.83^a	64.29±9.22^ab	78.57±9.22^ab	49.99±9.22^ab	28.57±10.10^a	14.29±5.83^a
B. bassiana AAUMB-21	14.28±10.10^b	53.57±6.83^ab	71.43±5.81^ab	32.14±12.19^ab	ND	ND
M. anisopliae AAUDM-43	7.14±7.14^b	71.43±11.66^a	82.14±8.99^ab	42.86±11.66^ab	21.43±9.22^ab	10.72±10.71^a
M. anisopliae AAUMFM-6	ND	32.14±12.19^bc	46.45±15.85^b	14.22±10.03^ab	ND	ND
Control	ND	ND	ND	ND	ND	ND

ND=not detected. Mean with Different letters in a column indicates the significant difference at Tukey’s HSD test, P < 0.05.

All taken together, the highest percentage colonization was displayed through the foliar application on the leaf (mean 73%), stem (mean 59%), and root (23%) compared to soil drenching with the highest percentage colonization on the root (mean 35%), stem (mean 16%) and leaf (8%). All isolates, except M. anisopliae AAUMFM-6, colonized root stem and leaf through the foliar spray. However, B. bassiana AAUMB-21 and M. anisopliae AAUMFM-6 failed to colonize stem and leaf through drenching (Table 3). Thus, B. bassiana AAUMB-29 was the most effective isolate with mean colonization of 50% on all parts through both methods of application, followed by B. bassiana AAUMFB-77 (48%) and M. anisopliae AAUDM-43 (39%).

In general, colonization rates of fungal isolates were significantly varied at the root (F=13.81, df=5,18, p<0.001), Stem (F=11.93, df=5,18, p=0.000) and Leaf (F=14.48, df=5,18, p=0.000) following foliar spray technique (Table 2). In addition, soil drenching inoculation technique verified significant variation in colonization rates of fungal strains on the root (F=3.93, df=5, 18, p=0.014), stem (F=6.08, df=5, 18, p<0.05) and leaf (F=2.04, df=5,18, p=0.122) of tomato plants (Table 2). It is interesting to note that all isolates showed 100% endophytic colonization of stem and leaf with foliar spray and root colonization in soil drenching method. The result also showed that B. bassiana isolates were more effective in colonizing tomato plants compared to M.anisopliae isolates.

Effects of entomopathogens on the control of Trialeurodes vaporariorum

The entomopathogenic fungal isolates of B. bassiana and M. anisopliae induced a significant reduction in the number of nymphs (F=51.72, df=5,24, p<0.001) and adults of T. vaporariorum (F=11.83, df=5,24, p<0.001) (Table 3). Thus, the application of B. bassiana AAUMB-29 reduced the nymphal population by 67% followed by M. anisopliaeAAUDM-43 and B. bassiana AAUMFB-77 with a reduction of 64% and 61%, respectively (Table 3). The pattern of reduction was different with the adult in that M. anisopliaeAAUDM-43 and B. bassiana AAUMB-29 reduced the population by 52% and 46%, respectively indicating that adults were more resistant than the nymphs (Table 3).

Table 3: B. bassiana and M. anisopliae isolates on nymphs and adults of T.vaporariorum after 30 days of foliar spray

Isolates	Number of T.vaporariorum after 30 days of spray
Isolates	No of Nymph/leaf	% reduction	No of Adult/leaf	% reduction over control
Control	26.8±1.28^a	-	20.60±1.63^a	-
B. bassiana AAUMB-29	8.8±0.73^c	67	11.20±0.66^bc	46
B. bassiana AAUMFB-77	10.4±0.81^c	61	13.80±1.24^bc	33
B. bassiana AAUMB-21	18.4±1.36^b	31	15.80±1.16^b	23
M. anisopliae AAUDM-43	9.6±0.50^c	64	10.00±0.71^c	52
M. anisopliae AAUMFM-6	16.8±1.65^b	37	14.60±0.81^bc	29

Mean with Different letters in a column indicates the significant difference at Tukey’s HSD test, P < 0.05.

Compatibility of selected endophytic B. bassiana and M. anisopliae strains with pesticide and neem extracts

Germination

The entomopathogenic fungi B. bassianaAAUMB-29, M. anisopliae AAUDM-43, and B. bassiana AAUMFB-77, were evaluated for their compatibility interaction with neem extracts and selected insecticides. The compatibility test showed significant variation in the resistance of the isolates to the different IPM components (different concentrations of Neem crude extract, Neem oil, and chemical insecticides). At recommended dose of every IPM component, the isolates showed pattern of conidial germination of 70-92% (Mean=83) (M. anisopliae AAUMD-43) (Table 4), 73-89% (Mean 81% (B. bassiana AAUMB-29) (Table 5), and 49-88% (Mean= 70) (B. bassiana AAUMB-77) (Table 6). In all cases, the isolates showed high conidial germination of >80% treated with Neem components, and with reduced conidial germination (43-69%) at the highest concentration of the chemical insecticides.

Table 4: The effects of neem extract and chemical pesticides on germination, radial growth, and sporulation of M. anisopliaeAAUDM-43.

Treatments	Concentration	Conidial Germination (%)	Radial growth (mm)	Sporulation (1x10⁷conidia/ml)	BI	Cl
AAUDM-43 (Control)	10⁷conidia/ml	96.67±0.33^a	43.67±2.96^a	8.33±0.33^a	-	-
Neem oil	1/2RD	95.33±0.88^a	42.67±1.76^ab	8.00±0.57^a	98.02	C
	RD	91.67±2.60^ab	33.67±1.20^abcd	7.00±0.58^ab	81.86	C
	2RD	89.67±1.76^ab	32.00±1.73^abcde	5.66±0.33^ab	72.93	C
Neem crude extract	1/2RD	92.33±1.45^ab	43.33±1.76^ab	8.33±0.33^a	99.18	C
	RD	87.33±1.45^ab	33.66±1.76^abcd	8.00±0.58^a	86.60	C
	2RD	73.00±2.30^c	24.00±5.03^cde	6.67±0.88^ab	67.81	C
Hunter 40 EC	1/2RD	88.33±0.88^ab	31.67±0.88^abcde	7.67±0.88^ab	82.82	C
	RD	83.67±2.02^b	30.33±1.45^bcde	6.00±1.15^ab	72.27	C
	2RD	44.67±3.17^d	19.67±1.76^e	3.66±1.20^b	42.89	MT
Ethiothoate 40% EC	1/2RD	90.67±2.33^ab	36.67±2.90^abc	5.00±0.57^ab	74.65	C
	RD	69.67±2.02^c	28.66±2.60^cde	4.67±1.20^ab	62.17	C
	2RD	47.67±1.85^d	22.00±4.04^de	3.96±1.56^ab	49.05	MT

Mean with Different letters in a column indicates the significant difference at Tukey’s HSD test, P < 0.05. BI=biological index, Cl =classification, C=Compatible, MT= moderately toxic, RD=recommended Dose.

Vegetative growth

The neem extract and chemical insecticides showed significant effects on the vegetative growth of endophytic entomopathogenic fungal isolates of M. anisopliaeAAUDM-43 (F=9.24;df=12,26; P<0.001) (Table 4), B. bassiana AAUMB‑29 (F=10.31;df=12,26; P<0.001) (Table 5) and B.
bassiana AAUMFB-77 (F=16.48; df=12,26; P<0.001) (Table 6). Isolate B. bassiana AAUMB-29 (20-31mm), B. bassiana AAUMFB-77(16-29 mm) and M. anisopliae AAUDM-43 (29-34mm) displayed better vegetative growth at the recommended dose of IPM components.

Spore production

The highest concentration of chemical pesticides and neem extracts had the most adverse effects on conidial production isolates compared to the lowest concentrations. The conidial production of isolates growing with insecticides indicated significance differences on M. anisopliae AAUDM-43 (F=7.56; df=12, 26; P<0.001) (Table 4), B.bassiana AAUMB-29 (F=2.32; df=12, 26; P=0.035) (Table 5). In contrast, both chemical insecticides and neem extracts were did not show significant variation on B. bassiana AAUMFB-77 (F=1.74; df=12, 26; P>0.05) (Table 6).

The maximum conidial yield was recorded from isolates without insecticides and neem extracts (untreated control). Generally, the highest concentration of chemical pesticides and neem extracts had the most adverse effects on germination, vegetative growth, and conidial production of endophytic fungal isolates compared to the lowest concentrations.

Table 5: The effects of neem extract and chemical pesticides on germination, radial growth,
and sporulation of B.bassiana AAUMB-29

Treatments	Concentration	Conidial Germination (%)	Radial growth (mm)	Sporulation (1x10⁷ conidia/ml)	BI	Cl
AAUMB-29 (Control)	10⁷conidia/ml	98.00±0.58^a	34.33±3.48^a	6.33±1.20^a	-	-
Neem oil	1/2RD	96.67±0.88^ab	33.00±2.65^ab	6.00±0.57^a	95.81	C
	RD	89.33±2.40^abc	31.00±3.60^abc	5.33±0.66^a	87.77	C
	2RD	85.00±2.52^abcd	28.33±1.85^abcd	5.00±0.57^a	81.42	C
Neem seed crude Extract	1/2RD	96.33±0.88^ab	32.66±1.85^ab	5.67±0.88^a	93.05	C
	RD	81.67±2.02^bcd	29.00±2.00^abcd	4.67±0.88^a	79.76	C
	2RD	72.67±5.36^d	24.00±1.00^abcd	4.00±0.58^a	67.44	C
Hunter 40 EC	1/2RD	91.67±2.72^ab	21.00±1.15^cde	4.33±0.88^a	67.51	C
	RD	81.00±1.52^bcd	19.67±0.88^de	4.00±1.26^a	62.85	C
	2RD	54.67±7.3^e	13.33±1.76^e	2.97±1.18^a	44.03	MT
Ethiothoate 40% EC	1/2RD	87.00±1.15^abcd	29.33±2.73^abcd	4.66±0.66^a	80.69	C
	RD	73.33±2.02^cd	22.00±1.15^bcde	3.30±1.23^a	60.02	C
	2RD	52.67±4.33^e	14.67±2.18^e	2.63±0.91^a	43.32	MT

Mean with Different letters in a column indicates the significant difference at Tukey’s HSD test, P < 0.05. BI=biological index, Cl =classification, C=Compatible, MT= moderately toxic, RD=recommended Dose.

Compatibility Evaluation

Based on the biological index (BI) and compatible classification (CL) entomopathogenic fungal isolates of B. bassiana AAUMB-29 and M. anisopliae AAUDM-43 were compatible to all components and concentrations except to the double doses (2RD) of Hunter 40 EC and Ethiothoate 40% EC that were classified as moderately toxic. On the contrary, B. bassiana AAUMFB-77 was compatible to all components except high doses (2RD) of Hunter 40 EC and Ethiothoate 40% EC and even to the same concentration of neem crude extract that was toxic to the tested isolates.

Table 6: The effects of neem extract and chemical pesticides on germination, radial growth, and sporulation of entomopathogenic fungi B.bassianaAAUMB-77

Treatments	Concentration	Conidial Germination (%)	Radial growth (mm)	Sporulation (1x10⁷ conidia/ml)	BI	Cl
AAUMFB-77 (Control)	10⁷conidia/ml	93.67±0.88^a	35.33±2.40^a	3.33±0.88^a	-	-
Neem oil	1/2RD	90.67±1.20^ab	30.00±1.52^ab	3.30±1.23^a	92.38	C
	RD	87.67±1.20^ab	29.33±1.20^ab	1.90±1.05^a	72.91	C
	2RD	85.67±3.17^ab	25.00±0.58^bc	1.80±0.76^a	65.65	C
Neem seed crude extract	1/2RD	90.00±0.57^ab	23.33±2.02^bcd	3.00±0.58^a	79.39	C
	RD	81.33±1.45^b	16.66±1.45^cde	2.30±0.91^a	61.09	C
	2RD	65.33±4.63^c	15.67±1.45^de	0.73±0.08^a	38.58	T
Hunter 40 EC	1/2RD	57.33±2.72^cd	28.00±1.73^ab	2.97±1.03^a	81.72	C
	RD	49.33±0.88^de	24.33±1.45^bc	2.60±0.95^a	71.17	C
	2RD	39.00±2.64^e	12.00±1.00^e	0.83±0.07^a	30.85	T
Ethiothoate 40% EC	1/2RD	80.67±2.72^b	28.67±2.40^ab	3.00±0.57^a	85.49	C
	RD	62.00±1.73^c	24.67±1.45^bc	1.66±0.33^a	70.27	C
	2RD	40.33±1.76^e	18.00±1.73^cde	0.67±0.18^a	36.90	T

Mean with Different letters in a column indicates the significant difference at Tukey’s HSD test, P < 0.05. BI=biological index, Cl =classification, C=Compatible, MT= moderately toxic, RD=recommended Dose.

Fungal entomopathogens as endophytes of wide ranges of plant species provide natural protection against various insect pests (Barra-Bucarei et al., 2020). The result of this particular study demonstrated that the foliar spray and soil drenching inoculation methods were successfully introduced to different strains of B. bassiana and M. anisopliae onto tomato plants. Thus, B. bassiana AAUMB-29 was the most effective isolate with mean colonization of 50% on all parts through both methods of application, followed by B. bassiana AAUMFB-77 (48%) and M. anisopliae AAUDM-43 (39%). These entomopathogenic fungal strains recovered from different parts of tomato plant tissues (roots, stems, and leaves) that confirmed their potential to move and establish throughout the plant parts. The entophytic colonization of tomato (Mwamburi, 2021; Silva et al., 2020), soybean (Russo et al., 2019), pecan (Ramakuwela et al., 2020), and coconut (Gaviria et al., 2020) have been achieved by both B. bassiana and M. anisopliae strains. This suggests that these entomopathogenic fungal isolates have the potential to colonize different plant species besides to tomatoes.

The colonization effects of roots, leaves, and stems of tomato plants were varied among B. bassiana and M. anisopliae strains. The B. bassiana entophytes dominantly colonized different parts of tomato plants as compared to M. anisopliae isolates. The B. bassiana has been reported endophytic establishment in the entire plant parts regardless of inoculation methods (Behie et al., 2015) in contrast M. anisopliae isolate was found to be preferentially localized inoculation sites (Liao et al., 2014). This result conceded with the finding of others suggesting that the inoculated entomopathogenic fungi penetrated plant epidermis into the leaf internal organs and move up and downward from inoculation sites and systematically colonized the entire plant parts (intercellular spaces and vascular xylem elements)(Bamisile et al., 2020; Quesada-Moraga et al., 2006).

In this result, the foliar spraying technique received the most effective colonization efficiency than the soil drenching method. Similarly, among leaf spray, soil drenching, and seed inoculation methods, the greater endophytic colonization efficiency of B. bassiana was obtained from the leaf spraying technique in tomato plants (Allegrucci et al., 2017; Pourtaghi et al., 2020). The endophytic colonization potential of entomopathogenic fungi on plants could be determined by the inoculation methods, fungal isolates, inoculum concentration, tissue specificity of fungal endophytes, environmental condition, and plant species (Afandhi et al., 2019; Qayyum et al., 2015). The efficacious colonization establishments of isolates by foliar spraying method could be due to the presence of stomata structures that provides an exchange channel in the different plant species.

Endophytic entomopathogenic fungi B. bassiana and M. anisopliae isolates were induced reduction in the number of nymphs (67%) and adults (52%) of T. vaporariorum over

un-inoculated treatments. The comparable result validated that B.bassiana achieved the population reduction of adults (52%) (Pourtaghi et al., 2020) and nymphs (65%) of T. vaporariorum from tomato (L. esculentum) (Barra-Bucarei et al., 2020). The significant reduction of nymphs and adults may relate to the production of secondary metabolites (bassiacridin, bassianolide, and beauvericin) by fungal entomopathogens (Rohlfs and Churchill, 2011) and plants in response to entomopathogenic fungal endophytes which has toxic, antifeedant, and repellent effects on insect pests (Shrivastava et al., 2015). In this result, the nymphal stages of T. vaporariorum were more susceptible to isolates of B. bassiana and M. anisopliae compared to adult stages. Likewise, (Mascarin et al., 2013) and (Barra-Bucarei et al., 2020) demonstrated that the nymphal stages of whitefly T. vaporariorum were more susceptible to endophytic entomopathogenic fungi B. bassiana and M. anisopliae. This may be due to the direct contact of conidial suspension of isolates on the surface of nymphs as a result of limited mobility and sticky natures of whitefly nymphs.

The compatibility test indicated significant variation in the resistance of entomopathogenic fungal isolates to the different concentrations of neem extract and chemical pesticides. Isolates of M. anisopliae AAUDM-43, B. bassianaAAUMFB-77, and B. bassianaAAUMB-29 displayed different levels of inhibition on their vegetative growth, germination, and sporulation in a response to all tested concentrations of neem extract and chemical pesticides. Correspondingly,

the chemical pesticides and plant extracts demonstrated variable inhibition on conidial germination (Abidin et al., 2017), vegetative growth (Abidin et al., 2017; Afandhi et al., 2020), and sporulation (Potrich et al., 2018; Yadav et al., 2019) of B. basianaand M. anisopliae strains.

Moreover, the study verified the variability level of interaction of chemical pesticides with entomopathogenic fungi such as M. anisolpiae and I. farinose (Borisade et al., 2016). Thus,

The highest concentration of chemical pesticides and neem extracts had the most adverse effects on germination, vegetative growth, and conidial production of isolates compared to the lowest concentrations. Moreover, all treatments were showed lower vegetative growth, spore germination, and conidial production in comparison to control treatments.

The compatibility evaluation based on Biological index formula analysis revealed that isolates of B. bassiana AAUMB-29, B. bassiana AAUMFB-77, and M. anisopliae AAUDM-43 were compatible with sub-lethal and lethal concentrations of neem extracts and chemical insecticides. Similarly, the entomopathogenic fungi were compatible with insecticides at sub-lethal concentrations and their compatible interaction increases pest control efficiency (Pelizza et al., 2018).

Entomopathogenic fungi B. bassiana and M. anisopliae colonized tomato plants and the foliar spraying technique was received as the most efficient inoculation technique. Isolates of B. bassiana and M. anisopliae isolates convinced a significant reduction in the number of nymphs and adults stages of T. vaporariorum. The nymphal stages of T. vaporariorum were more susceptible to both isolates of B. bassiana and M. anisopliae compared to adult stages. The in-vitro compatibility study showed that sub-lethal and lethal concentrations of tested neem extracts and chemical insecticides were compatible with both B. bassiana and M. anisopliae isolates. The endophytic B.bassiana AAUMB-29, and M. anisopliae AAUDM-43, significantly reduced the population of T. vaporariorum, and were highly compatible with selected pesticides. The combined use of B. bassiana AAUMB-29 and M. anisopliae AAUDM-43 with neem oil and insecticide (Hunter 40 EC) at concentrations lower than the field-recommended dose should be considered in the integrated management of T. vaporariorum and other insect pests.

Ethics approval and consent to participate

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Consent for publication

Not applicable

Availability of data and materials

All data and material are stated in the manuscript

Competing interests

No potential conflict of interest reported by authors

Funding

Culture media, Chemicals, reagents, substrates, stationary materials, and laboratory and field equipment were supplied by Ethiopian Biotechnology Institute and the Regional Project.

Authors' contributions

AG: planned the study, carried out the experiment, analyzed data, and wrote the manuscript. EM: participated in laboratory experiments. FA: supervised the overall activities in the study and editing manuscript. All authors read and approved the manuscript.

Acknowledgments

The authors gratefully thank the Healthy Seedling Project granted by both the Ethiopian Biotechnology Institute and the Regional Project supported by the Austrian Development Agency (ADA) for financial support in this study.

Abidin, A. F., Ekowati, N., and Ratnaningtyas, N. I. (2017). Compatibility of insecticides with entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. Scripta Biologica 4, 273-279.
Afandhi, A., Pratiwi, V. R., Hadi, M. S., Setiawan, Y., and Puspitarini, R. D. (2020). Suitable Combination Between Beauveria bassiana (Balsamo) Vuillemin and Four Plant Leaf Extracts to Control Spodoptera litura (Fabricius). Agrivita 42, 341.
Afandhi, A., Widjayanti, T., Emi, A. A. L., Tarno, H., Afiyanti, M., and Handoko, R. N. S. (2019). Endophytic fungi Beauveria bassiana Balsamo accelerates growth of common bean (Phaeseolus vulgaris L.). Chemical and Biological Technologies in Agriculture 6, 11.
Ali, S., Farooqi, M. A., Sajjad, A., Ullah, M. I., Qureshi, A. K., Siddique, B., Waheed, W., Sarfraz, M., and Asghar, A. (2018). Compatibility of entomopathogenic fungi and botanical extracts against the wheat aphid, Sitobion avenae (Fab.)(Hemiptera: Aphididae). Egyptian Journal of Biological Pest Control 28, 1-6.
Allegrucci, N., Velazquez, M. S., Russo, M. L., Pérez, M. E., and Scorsetti, A. C. (2017). Endophytic colonisation of tomato by the entomopathogenic fungus Beauveria bassiana: the use of different inoculation techniques and their effects on the tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae).
Alves, S., Haddad, M., Faion, M., Baptista, G., and Rossi-Zalaf, L. (2007). Novo índice biológico para a classificação de agrotóxicos para fungos entomopatogênicos. Resumos, 10th Simpósio de Controle Biológico. Brasília, Brasil.
Bamisile, B. S., Dash, C. K., Akutse, K. S., Keppanan, R., Afolabi, O. G., Hussain, M., Qasim, M., and Wang, L. (2018). Prospects of endophytic fungal entomopathogens as biocontrol and plant growth promoting agents: An insight on how artificial inoculation methods affect endophytic colonization of host plants. Microbiological Research 217, 34-50.
Bamisile, B. S., Senyo Akutse, K., Dash, C. K., Qasim, M., Ramos Aguila, L. C., Ashraf, H. J., Huang, W., Hussain, M., Chen, S., and Wang, L. (2020). Effects of seedling age on colonization patterns of Citrus limon plants by endophytic Beauveria bassiana and Metarhizium anisopliae and their influence on seedlings growth. Journal of Fungi 6, 29.
Barra-Bucarei, L., González, M. G., Iglesias, A. F., Aguayo, G. S., Peñalosa, M. G., and Vera, P. V. (2020). Beauveria bassiana multifunction as an endophyte: growth promotion and biologic control of Trialeurodes vaporariorum,(Westwood)(Hemiptera: Aleyrodidae) in tomato. Insects 11, 591.
Behie, S. W., Jones, S. J., and Bidochka, M. J. (2015). Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria. Fungal Ecology 13, 112-119.
Borisade, O., Oso, A., and Falade, M. (2016). Interactions of some registered agrochemicals in Nigerian farming systems with entomopathogenic fungi, Metarhizium anisopliae and Isaria farinose. Ife Journal of Science 18, 949-961.
Brunner-Mendoza, C., Reyes-Montes, M. d. R., Moonjely, S., Bidochka, M. J., and Toriello, C. (2019). A review on the genus Metarhizium as an entomopathogenic microbial biocontrol agent with emphasis on its use and utility in Mexico. Biocontrol Science and Technology 29, 83-102.
Chi, M.-H., Park, S.-Y., and Lee, Y.-H. (2009). A quick and safe method for fungal DNA extraction. The Plant Pathology Journal 25, 108-111.
Chonoko, U., and Rufai, A. (2011). Phytochemical screening and antibacterial activity of Cucurbita pepo (Pumpkin) against Staphylococcus aureus and Salmonella typhi. Bayero Journal of Pure and Applied Sciences 4, 145-147.
Gaviria, J., Parra, P. P., and Gonzales, A. (2020). Selection of strains of Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales) for endophytic colonization in coconut seedlings. Chilean journal of agricultural & animal sciences 36, 3-13.
Jaber, L. R., and Ownley, B. H. (2018). Can we use entomopathogenic fungi as endophytes for dual biological control of insect pests and plant pathogens? Biological control 116, 36-45.
Jha, S. K., and Kumar, M. (2017). Relative efficacy of different insecticides against whitefly, Bemisia tabaci on tomato under field condition. Journal of Entomology and Zoology Studies 5, 728-732.
Kabaluk, T., Goettel, M., Ericsson, J., Erlandson, M., Cassidy, F., Vernon, B., Jaronski, S., Mackenzie, K., and Cosgrove, L. (2007). Promise versus performance: Working toward the use of Metarhizium anisopliae as a biological control for wireworms. IOBC WPRS BULLETIN 30, 69.
Kebede, Y., Gebre-Michael, T., and Balkew, M. (2010). Laboratory and field evaluation of neem (Azadirachta indica A. Juss) and Chinaberry (Melia azedarach L.) oils as repellents against Phlebotomus orientalis and P. bergeroti (Diptera: Psychodidae) in Ethiopia. Acta tropica 113, 145-150.
Kim, J. J., Jeong, G., Han, J. H., and Lee, S. (2013). Biological control of aphid using fungal culture and culture filtrates of Beauveria bassiana. Mycobiology 41, 221-224.
Lacey, L. A. (2012). "Manual of techniques in invertebrate pathology," Academic Press.
Liao, X., O’Brien, T. R., Fang, W., and Leger, R. J. S. (2014). The plant beneficial effects of Metarhizium species correlate with their association with roots. Applied microbiology and biotechnology 98, 7089-7096.
Maina, U., Galadima, I., Gambo, F., and Zakaria, D. (2018). A review on the use of entomopathogenic fungi in the management of insect pests of field crops. Journal of Entomology and Zoology Studies 6, 27-32.
Martin, J. H., Mifsud, D., and Rapisarda, C. (2000). The whiteflies (Hemiptera: Aleyrodidae) of Europe and the Mediterranean basin. Bulletin of entomological research 90, 407-448.
Mascarin, G. M., Kobori, N. N., Quintela, E. D., and Delalibera Jr, I. (2013). The virulence of entomopathogenic fungi against Bemisia tabaci biotype B (Hemiptera: Aleyrodidae) and their conidial production using solid substrate fermentation. Biological Control 66, 209-218.
Meyling, N. V., and Eilenberg, J. (2006). Occurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem. Agriculture, ecosystems & environment 113, 336-341.
Moorhouse, E., Gillespie, A., Sellers, E., and Charnley, A. (1992). Influence of fungicides and insecticides on the entomogenous fungus Metarhizium anisopliae a pathogen of the vine weevil, Otiorhynchus sulcatus. Biocontrol Science and Technology 2, 49-58.
Mora, M. A. E., Castilho, A. M. C., and Fraga, M. E. (2017). Classification and infection mechanism of entomopathogenic fungi. Arquivos do Instituto Biológico 84.
Mustafa, U., and Kaur, G. (2009). Effects of carbon and nitrogen sources and ratio on the germination, growth and sporulation characteristics of Metarhizium anisopliae and Beauveria bassiana isolates. African Journal of Agricultural Research 4, 922-930.
Mwamburi, L. A. (2021). Endophytic fungi, Beauveria bassiana and Metarhizium anisopliae, confer control of the fall armyworm, Spodoptera frugiperda (JE Smith)(Lepidoptera: Noctuidae), in two tomato varieties. Egyptian Journal of Biological Pest Control 31, 1-6.
Navas-Castillo, J., Fiallo-Olivé, E., and Sánchez-Campos, S. (2011). Emerging virus diseases transmitted by whiteflies. Annual review of phytopathology 49, 219-248.
Parsa, S., García-Lemos, A. M., Castillo, K., Ortiz, V., López-Lavalle, L. A. B., Braun, J., and Vega, F. E. (2016). Fungal endophytes in germinated seeds of the common bean, Phaseolus vulgaris. Fungal biology 120, 783-790.
Pelizza, S. A., Schalamuk, S., Simón, M. R., Stenglein, S. A., Pacheco-Marino, S. G., and Scorsetti, A. C. (2018). Compatibility of chemical insecticides and entomopathogenic fungi for control of soybean defoliating pest, Rachiplusia nu. Revista Argentina de Microbiología 50, 189-201.
Petrini, O., and Fisher, P. (1986). Fungal endophytes in Salicornia perennis. Transactions of the British Mycological Society 87, 647-651.
Posada, F., Aime, M. C., Peterson, S. W., Rehner, S. A., and Vega, F. E. (2007). Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycological research 111, 748-757.
Potrich, M., Dallacort, S., Luckman, D., Lozano, E., Pegorini, C., and Simionatto, D. (2018). Compatibility of Beauveria bassiana and alternative phytosanitary products. Journal of applied microbiology 125, 1802-1811.
Pourtaghi, E., Talaei-Hassanloui, R., Nasibi, F., and Fotouhifar, K.-B. (2020). Endophytic colonization of tomato by Beauveria bassiana for control of the greenhouse whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). Acta Biologica 27, 149-160.
Qayyum, M. A., Wakil, W., Arif, M. J., Sahi, S. T., and Dunlap, C. A. (2015). Infection of Helicoverpa armigera by endophytic Beauveria bassiana colonizing tomato plants. Biological Control 90, 200-207.
Quesada-Moraga, E., Landa, B., Muñoz-Ledesma, J., Jiménez-Diáz, R., and Santiago-Alvarez, C. (2006). Endophytic colonisation of opium poppy, Papaver somniferum, by an entomopathogenic Beauveria bassiana strain. Mycopathologia 161, 323-329.
Ramakuwela, T., Hatting, J., Bock, C., Vega, F. E., Wells, L., Mbata, G. N., and Shapiro-Ilan, D. (2020). Establishment of Beauveria bassiana as a fungal endophyte in pecan (Carya illinoinensis) seedlings and its virulence against pecan insect pests. Biological Control 140, 104102.
Rohlfs, M., and Churchill, A. C. (2011). Fungal secondary metabolites as modulators of interactions with insects and other arthropods. Fungal Genetics and Biology 48, 23-34.
Russo, M. L., Pelizza, S. A., Vianna, M. F., Allegrucci, N., Cabello, M. N., Toledo, A. V., Mourelos, C., and Scorsetti, A. C. (2019). Effect of endophytic entomopathogenic fungi on soybean Glycine max (L.) Merr. growth and yield. Journal of King Saud University-Science 31, 728-736.
Shoukat, R. F., Freed, S., and Ahmad, K. W. (2016). Evaluation of binary mixtures of entomogenous fungi and botanicals on biological parameters of Culex pipiens (Diptera: Culicidae) under laboratory and field conditions. Int. J. Mosq. Res 3, 17-24.
Shrivastava, G., Ownley, B. H., Augé, R. M., Toler, H., Dee, M., Vu, A., Köllner, T. G., and Chen, F. (2015). Colonization by arbuscular mycorrhizal and endophytic fungi enhanced terpene production in tomato plants and their defense against a herbivorous insect. Symbiosis 65, 65-74.
Silva, A. C. L., Silva, G. A., Abib, P. H. N., Carolino, A. T., and Samuels, R. I. (2020). Endophytic colonization of tomato plants by the entomopathogenic fungus Beauveria bassiana for controlling the South American tomato pinworm, Tuta absoluta. CABI Agriculture and Bioscience 1, 1-9.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution 24, 1596-1599.
Vasantharaj, S., Sathiyavimal, S., and Hemashenpagam, N. (2013). Phytochemical analysis and antibacterial activity of Azadirachta indica. International Journal of Pharmaceutical Sciences Review and Research 22, 59-61.
Vidal, S., and Jaber, L. R. (2015). Entomopathogenic fungi as endophytes: plant–endophyte–herbivore interactions and prospects for use in biological control. Current science, 46-54.
White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18, 315-322.
Wu, S., Toews, M. D., Oliveira-Hofman, C., Behle, R. W., Simmons, A. M., and Shapiro-Ilan, D. I. (2020). Environmental tolerance of entomopathogenic fungi: A new strain of Cordyceps javanica isolated from a whitefly epizootic versus commercial fungal strains. Insects 11, 711.
Yadav, D. S., Ranade, Y., Mhaske, S., and Ghule, S. (2019). Compatibility of insecticides with Metarhizium brunneum (Petch) and Beauveria bassiana (Bals.) for bio-intensive management of pink mealybug, Maconellicoccus hirsutus (Green) in grapes. Journal of Biological Control 33, 253-263.
Yii, J. E., Bong, C. F. J., King, J. H. P., and Kadir, J. (2015). Synergism of entomopathogenic fungus, Metarhizium anisopliae incorporated with fipronil against oil palm pest subterranean termite, Coptotermes curvignathus. Plant Protection Science 52, 35-44.
Zafar, J., Freed, S., Khan, B. A., and Farooq, M. (2016a). Effectiveness of Beauveria bassiana against cotton whitefly, Bemisia tabaci (Gennadius)(Aleyrodidae: Homoptera) on different host plants. Pak. J. Zool 48, 91-99.
Zafar, J., Freed, S., Khan, B. A., and Farooq, M. (2016b). Effectiveness of Beauveria bassiana against cotton whitefly, Bemisia tabaci (Gennadius)(Aleyrodidae: Homoptera) on different host plants. Pakistan Journal of Zoology 48.
Zhang, C., SHAO, Z.-f., HAN, Y.-y., WANG, X.-m., WANG, Z.-q., Musa, P. D., QIU, B.-l., and Ali, S. (2018). Effects of Aschersonia aleyrodis on the life table and demographic parameters of Bemisia tabaci. Journal of integrative agriculture 17, 389-396.

Effects of Myco-Entomopathogens on Bio-Control of Trialeurodes Vaporariorum, Endophytic Colonization of Tomato, and Compatibility with selected pesticides

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Abstract

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Introduction

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