Effectiveness some Egyptian isolates of entomopathogenic fungi against bulb mite (Rhizoglyphus robini - Acaridae)

doi:10.21203/rs.3.rs-2468795/v1

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Effectiveness some Egyptian isolates of entomopathogenic fungi against bulb mite (Rhizoglyphus robini - Acaridae)

https://doi.org/10.21203/rs.3.rs-2468795/v1

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Five fungal isolates have been evaluated from the Egyptian environment for activity against Rhizoglyphus robini; these isolates were Aspergillus terreus1, Aspergillus terreus2, Aspergillus flavus, Myrothecium advena, and Penicillin crustostum. These isolates were evaluated by different concentrations. The concentrations 3.2x10⁶ conidia/ml for A.terreus1was effective against tested mite than M. advena, A. terreus 2, A. flavus, and P. crustostum. The mortality percentages were (78.62, 69.66, 55.86, 17.93, and 1.38%); The LC₅₀ values exhibited by A. terrus1, M. advena, A. terreus2 were (1.06x10⁶, 1.75x10⁶, 3.45x10⁶, and 7.4x10⁹) conidia /ml, respectively.

entomopathogenic fungi

Aspergillus terreus

Myrothecium advena

The bulb mite, Rhizoglyphus robini Claparède (Acari: Acaridae), is considered one of the most destructive pests of onion, garlic, and ornamentals such as lily, tulips, and hyacinths(Diaz, Okabe et al., 2000). As is the case with other mites in the family Acaridae, Rhizoglyphus mites are generally understudied and are often overlooked by non-specialists. Even though new species are frequently described (McCoy, Samson et al., 1988). Bulb mites are often intercepted in commercial shipments of infested crops(Manson 1972)., particularly R. robini and R. echinopus. Dispersal of Rhizoglyphus mites due to human activities further complicates our understanding of their biogeography. Bulb mites that establish in the inner layers are difficult to control. Infested bulbs show reddish-brown discoloration on the fleshy scales and frequently rot before or after planting. Should the infested bulb grow, the leaves will be stunted, distorted and soon turn yellow. The flower stalk will be destroyed or will fail to develop, in storage at temperatures above 40°C; the mite will continue to multiply and migrate to other bulbs, thus infesting the entire stored lot. Lightly infested bulbs held in cold storage below 40°C may still become seriously infested during shipment, store shelves, or after planting, whenever temperature conditions become favorable. Thus the search for environmentally safe alternative control strategies is of paramount importance. Biopesticides are pesticides are derived from organisms such as animals, plants, or microorganisms. They can be natural enemies or secondary metabolites of these organisms such as phytochemicals and microbial products(Canan 2013).There is increasing attention toward using biopesticides to develop a safe, eco-friendly and integrated pest management approach to control pests(Copping and Menn 2000). Microbial-Control based pesticides form the bulk of biopesticides in the market since; there are approximately 175 microbial biopesticides used to control insects, pathogens, nematodes, and weeds(Lengai, Muthomi et al. 2018). Fungal genera include Trichoderma, Beauveria, Metarhizium, and Paecilomyces are used in biopesticides(Kachhawa and Studies 2017). Efforts to develop biocontrol techniques for bulb mites have been undertaken in many countries and mainly involved using soil-dwelling predatory mites, Hypoaspis aculeifer (Acari: Laelapidae) (Lesna, Sabelis et al. 1996). Many species of entomopathogenic fungi attack Acari and can be used for biological Control of mite pests(Chandler, Davidson et al. 2000). Such as Metarhizium spp. and Beauveria spp. are known to inhibit the growth and reproduction of a wide range of herbivores from different feeding guilds. These include members of the Coleoptera, Diptera, Lepidoptera, and Orthoptera (Bamisile, Dash et al. 2018, Gange, Koricheva et al. 2019)., But this option is now limited mainly due to the withdrawal of some chemical pesticides. Thus, alternative control methods, biological control need to be developed and implemented. Besides using predatory mites, entomopathogenic fungi (EPF) might be prospective biocontrol agents against R. robini. Our study's objectives were to determine the pathogenicity and culture filtrate of some strains of EPFs to the bulb mite and evaluate their potential for its biological Control.

1-Isolation, purification, and identification of entomopathogenic fungi:

Dead insects were selected for the present investigation, which was collected from their natural habitats. The surface of insects were sterilized using ethanol 70% (v/v) for 30 s, followed by 1% (v/v) sodium hypochlorite solution (NaOCl) for 30 s, 70% (v/v) ethanol for 30 s, and rinsing with distilled water 2–3 times(Sopalun, Strobel, et al. 2003) then dried using sterile filter paper. Sterile pest cadavers were placed on potato dextrose agar (PDA) in Petri dishes and incubated at 25 ± 1°C. After the emergence of fungal hyphae and sporulation, they were subcultured by transferring onto a new PDA plate and incubated at 25°C. The purified entomopathogenic fungi isolates were transferred separately into PDA slants and maintained at 4 ºC till further studies.

2-Identification of entomopathogenic fungi by morphological methods:

Entomopathogenic fungi isolates were identified at Assiut University Moubasher Mycological Center (AUMMC) Assiut, Egypt, based on their macro-and microscopic characteristics as described previously (Raper and Thom 1949, Ellis 1968, Domsch, Gams et al. 1980, Bennett and genomics 2010).

3- Preparation of the fungal conidial concentrations.

Spores of fungal isolates harvested by rinsing with sterilized water, 0.5% Tween 80 from 14 day culture media (PDA). The suspensions were filtered through a cheese cloth to reduce mycelium clumping. The spores were counted in the suspension using a hemocytometer (0.1 mm x 0.0025 mm2). Five concentrations were prepared, (C1) 3.2x10⁶, (C2) 2.4x10⁶, (C3) 1.6x10⁶, (C4) 0.8 x10⁶and (C5) 0.4 x10⁶ Spore/ml in all isolates(Abdel-Raheem, ALghamdi et al. 2019).

4- Mites Culturing:

Stock cultures of R. robini were established from infested samples of onion reared in a plastic chamber (6 cm in diameter× 1.5 cm thick) filled partially with a mixture cement: clay: charcoal (7: 2: 1). These plastic chambers contain dried onions, granules, and few drops of water as sources of food and humidity, covered with soft plastic cover after adding two females and males from the laboratory culture mite. These cultures were observed daily and kept in an incubator at 25 C and 70% R.H for the biocontrol experiment.

5- Bioassay of conidial suspension against Rhizoglyphus robini adult female:

The sterilized filter paper was placed in 55 mm diameter Petri dishes and impregnated with 0.5 mL sterile water. Adult mites (30 per dish) were placed on the impregnated filter paper with a camel-hair brush, fungus (A. terreus1, A. terreus2, A. flavus, P. crustostum and Myrothecium advena) were sprayed using a manual sprayer in a suspension containing 3.2 x 10⁶, 2.4 x 10⁶, 1.6 x 10⁶, 0.8 x 10⁶ and 0.4 x 10⁶ spore/ml; Control were treated with distilled water with 0.5% Tween 80. The number of infected mites (i.e., mites on which mycelia developed) was counted after 3, 5, 7, and 9 days under a stereomicroscope. Each treatment contained five replicates. Adult mortality was evaluated until the end of the experiment, and the mortality was corrected using Abbott's formula (Abbott 1925), Lethal concentrations (LC₅₀ and LC₉₀) were estimated by Probity analysis(Aitchison and Silvey 1957).

1-Isolation and identification of entomopathogenic fungi:

Isolation of the entomopathogenic fungi was carried out under aseptic conditions. Data exhibited that entomopathogenic fungi were isolated and found in association with dead insects. (Table 1) that five isolates of entomopathogenic fungi were isolated from dead insects and identified based on their macro and microscopic characteristics (Fig. 1). These isolates were; A flavus, A terreus1, A terreus2, M advena, and P. crustostum.

2- Effect of A. terreus 1, A. terreus 2, A. flavus, M. advena and p. crustostum conidial on adult females of R. robini.

Results in (Table 2) showed a concentration-dependent effectiveness; Data presented show cumulative mortality percentages of R. robini adults were recorded after 9 days for treatments the highest percentages were (78.62, 62.76, 51.72, 39.31and 35.17%) for A. terreus 1 at the tested concentrations of (3.2 x 10⁶, 2.4 x 10⁶,1.6x 10⁶,0.8x 10⁶ and 0.4 x 10⁶ conidia/ml respectively, followed by the second highest mortality rate it is M. advena at the tested previous concentrations the mortality percentages were (69.66, 55.86, 46.90, 31.72 and 20.00%). The species A. terreus 2 with mortality percentages (55.86, 37.93, 31.72, 20.69, and 17.24%) while A. flavus and p. crustostum the mortality rates were (17.93, 16.55, 13.79, 11.72 and 11.03%) and (1.3, 0.69, 0.69, 0.00, and 0.00%) for the previous concentrations respectively and Comparing the previous results, we find that the highest mortality rate caused by both A. terreus 1, M. advena and A. terreus 2 (Fig. 2). The results also indicate that there are no effects for p. crustostum on R. robini. Mortalities occurred on the 3rd day and increased gradually and reached the maximum on the 9th day from treatment with all concentrations, the percent of mortalities increased with an increase of concentrations. The percent of mortalities ranged between 35.00 to 78% with A. terrus1, 20.00 to 69.66% with M. advena, and 17.24 to 55.86 with A. terrus1 9th day after treatment. The statistical analysis shows that there are significant differences between all concentrations in both isolations.

LC₅₀ and LC₉₀ values confirmed that R. robini was more susceptible to A. terreus1, M. advena, and A. terreus2 than A. flavus, LC₅₀ were (1.06 x10⁶,1.75 x10⁶, 3.45 x10⁶ and 7.42 x10⁹ conidia /ml) respectively. While LC₉₀ were (2.44 x10⁷, 2.83 x10⁷,4.5 x10⁷ and 6.83x10¹⁴ conidia /ml)respectively. The LT₅₀ was calculated as (6.77, 6.62, 8.09 and18.16 days) to the four fungi respective, indicating the superiority of A. terreus 1, M. advena over A. terreus2 and A. flavus Table (3).

Many researchers have documented the natural occurrence of EPF, as mention in the review by Zimmermann(Zimmermann and Technology 2007a), Zimmermann and Technology 2007 b). About 90 genera comprise more than 700 species of fungi that are pathogenic to insects (Thacker 2002, Lacey and Kaya 2007, Khachatourians and Qazi 2008, Hemasree 2013).,

Data are presented as mean value (no. of dead mites) ± SE. a, b Different letters indicate a significant difference according to Duncan’s multiple range tests. Values are significant at p = 0.05 levels, df = 129, F-value = 56.39 (after 9 days).

however, only a few species have been deeply studied for insect pest control in agriculture(Khan and Ahmad 2015), EPF were used against of insect and mite pests(Hajek 2005, Hajek, McManus et al. 2007), One of the most important of these genus representing a wide community of entomophorales Beauveria spp., Aspergillus spp., Fusarium spp., and Verticillium spp.(Ercan 2019). Moreover, due to Acari generally have many of them inhabit environments characterized with humid microclimates(Ferro and Southwick 1984, Evans 1992), they considered favorable hosts for fungal infections because these characteristics favor infection anding soft bodies. Consequently, EPF represents an efficient candidate as a biocontrol agent that plays a crucial role in controlling R. robini. The efficacy of A. niger and A. flavus was evaluated against red spider mites in the laboratory using three different concentrations of conidia (1x10⁶, 1x10⁷ and 1x10⁸ conidia/ml) at different interval of time. Results showed that both the isolates were effective against Oligonychus coffeae populations. However, efficacy of A. niger was much higher than A. flavus it was found that mortality percentage increased with the increase in conidial concentration. The highest mortality of 91.11% was recorded after 96 hours of treatment with A. niger at a concentration of 1x108 conidia/ml. A. flavus resulted in mortality percentage as high as 62.22% after 96 hours of treatment with the highest concentration(Mazid, Rajkhowa et al. 2015). Many authors demonstrated evaluated of acaricides potency of P. lilacinum isolate efficacy at spore concentration of 1.6× 10⁸ and 5× 10⁷conidia/ml, mortality rates were 77.97% and 71.19%, respectively, after 10 days. While after 7days, it killed only 67.34 and 57.24%, respectively., The median lethal concentration of P. lilacinum, is 2.85×106 conidia/ml on T. urticae (Nawar, Abo-Elnasr et al. 2018). On the other hand, seventeen isolates of Metarhizium anisopliae and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi. In the laboratory, all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose-response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7 × 10⁷ and 2.5 × 10⁷ conidia /ml. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days(Wekesa, Maniania et al. 2005).

As found in another study, the mite mortality rates increased with increasing conidial concentrations and days after treatment. The mortality rate caused by M. anisopliae isolates varied from 18.75 to 85%, with LC₅₀ value of 4.6 × 10⁵ conidia/ml and LC₉₀ value of 2.4 × 10⁸ conidia/ml during 7 days, respectively. The isolate of B. bassiana caused 15 to 70% mortality, and its LC₅₀ and LC₉₀ values estimated 3.3 × 10⁶ and 7.8 × 10⁹ conidia/ml, respectively. However, V. lecanii isolate caused 11.25 to 72.50% mortality with LC₅₀ of 5.2 × 10⁶ conidia/ml, while T. harzianum was potentially less virulent than other isolates causing 8.75 to 63.75% mortality rate to T. urticae with LC₅₀ of 9.4 × 10⁶ conidia/ml.(Elhakim, Mohamed et al. 2020), In addition to eleven strains of the genus Metarhizium sp., five strains of the genus Beauveria sp., 2 strains of the genus Isaria sp. and 2 strains of genus Lecanicillium sp. isolated from onions and garlic fields in Israel and the Czech Republic were tested against female R. robini females. The highest mortality (98–99%) was caused by Metarhizium sp(Zemek, Nermuť et al. 2020). Also A study on 12 entomopathogenic fungi for controlling broad mite Polyphagotarsonemus latus Banks in mulberry found that M. anisopliae CKM-048 was the most virulent strain in controlling both larvae and broad adult mites at the concentration of 2 x 10⁸ conidia/ml. There was no ovicidal effect when tested with broad mite eggs. Median lethal concentrations (LC₅₀of M. anisopliae in killing larvae and adults were 8.7 x 10⁶ and 1.3 x 10⁷ conidia/ml, respectively. Median lethal times LT₅₀ of larvae and adults were 2.4 and 3.8 days, respectively, at the concentration of 2 x 10⁸ conidia/ml.(Maketon, Orosz-Coghlan et al. 2008). Evaluation of the conidial potency of A. terrus1and M. advena isolates obtained in this study showed high efficacy at spore concentration of 3.2× 10⁶ conidia/ml. they exhibited mortality rates of 78.62% and 69.66 respectively but at spore concentration of 2.4× 10⁶ were 62.76 and 55.86% after 9 days respectively. while A. flavus showed low mortality reached and 17.93 and 16.55, Median lethal concentration of A. terrus1and M. advena is 1.06×10⁶ and 1.75 ×10⁶ conidia/ml, which was lower than values obtained by some Aspergillus sp isolates when it was tried on Oligonychus coffeae (Mazid, Rajkhowa et al. 2015).

Author Contributions: Author Contributions: Conceptualization, A.S. M. Mohamed methodology, A.S. M. Mohamed, Momamed Gamal Hezazy; statistical analysis,data curation. A.S. M. Mohamed, Liande Wang; writing—original draft preparation,writing—review and editing and All authors have read and agreed to the published version of the manuscript.

Acknowledgments: Thanks to all field technicians at Faculty of Agriculture, Al-Azhar University branch Assiut, Egypt.

Compliance with Ethical Standards This article does not contain any studies with human participants or animals performed by any of the authors.

Funding: This research received no external funding.

Data Availability Statements All data available within the article.

Consent for publication: Not applicable

Conflict of interest: The authors declare that they have no conflict of interest.

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Table (1): Isolation and identification of entomopathogenic fungi from insects.

AUMMC No.	Identification	Localities	Host
14530	Aspergillus flavus Link	Qena	Sitophilus granarius
14531	Aspergillus terreus (1) Thom	Qena	Sitophilus granarius
14532	Aspergillus terreus (2) Thom	Assiut	Cryptostigmata mite
14533	Myrothecium advena Sacc.	Assiut	Cryptostigmata mite
14534	Penicillium crustosum Thom	Qena	Sitophilus granarius

Table (2) Cumulative mortality percentages of R. robini mite treated with entomopathogenic fungi at different concentrations.

Fungi	AUMMC No	Conc. spores/ml.	Mean No. of dead adult of R. robini mites indicated days				% mortality	% Corrected mortality
Fungi	AUMMC No	Conc. spores/ml.	3	5	7	9	% mortality	% Corrected mortality
Aspergillus terreus 1	14531	3.2 x 106	0.80 ± 0.37	7.20 ± 0.86	14.20 ± 0.58	23.80 ± 0.73	79.33	78.62 ^a
		2.4 x 106	0.60 ± 0.24	4.80 ± 0.86	11.60 ± 0.81	19.20 ± 1.46	64.00	62.76^bc
		1.6 x 106	0.40 ± 0.40	3.20 ± 0.80	9.60 ± 1.43	16.00 ± 1.64	53.33	51.72^d
		0.8 x 106	0.40 ± 0.24	3.00 ± 0.63	7.40 ± 0.87	12.40 ± 1.36	41.33	39.31^ef
		0.4 x 106	0.20 ± 0.20	2.40 ± 0.87	4.40 ± 1.02	11.20 ± 0.91	37.33	35.17^f
Aspergillus terreus2	14532	3.2 x 106	0.60 ± 0.24	5.40 ± 0.92	11.80 ± 0.80	17.20 ± 0.73	57.33	55.86^cd
		2.4 x 106	0.40 ± 0.24	3.20 ± 0.37	7.80 ± 0.37	12.00 ± 0.70	40.00	37.93^ef
		1.6 x 106	0.40 ± 0.24	2.60 ± 0.67	6.20 ± 0.66	10.20 ± 0.73	34.00	31.72^f
		0.8 x 106	0.40 ± 0.24	2.40 ± 0.40	5.00 ± 0.89	7.00 ± 0.73	23.33	20.69^g
		0.4 x 106	0.20 ± 0.20	1.60 ± 0.50	3.80 ± 0.66	6.00 ± 0.83	20.00	17.24^g
Aspergillus flavus	14530	3.2 x 106	0.40 ± 0.24	3.40 ± 0.50	4.80 ± 0.48	6.20 ± 0.73	20.67	17.93^g
		2.4 x 106	0.20 ± 0.20	2.60 ± 0.24	4.60 ± 0.50	5.80 ± 0.37	19.33	16.55^g
		1.6 x 106	0.40 ± 0.24	2.20 ± 0.58	3.20 ± 0.66	5.00 ± 0.54	16.67	13.79^g
		0.8 x 106	0.20 ± 0.20	1.60 ± 0.50	2.80 ± 0.37	4.40 ± 0.50	14.67	11.72^g
		0.4 x 106	0.20 ± 0.20	1.20 ± 0.37	2.40 ± 0.50	4.20 ± 0.58	14.00	11.03^g
Myrothecium advena	14533	3.2 x 106	0.60 ± 0.40	8.40 ± 0.81	16.00 ± 0.54	21.20 ± 0.73	70.67	69.66^b
		2.4 x 106	0.20 ± 0.20	7.20 ± 0.58	13.20 ± 0.58	17.20 ± 0.80	57.33	55.86^cd
		1.6 x 106	0.40 ± 0.24	5.20 ± 1.01	11.00 ± 1.81	14.60 ± 1.53	48.67	46.90^de
		0.8 x 106	0.20 ± 0.20	4.20 ± 0.86	6.80 ± 1.15	10.20 ± 1.01	34.00	31.72^f
		0.4 x 106	0.20 ± 0.20	1.80 ± 0.68	3.00 ± 1.34	6.80 ± 1.71	22.67	20.00^g
penicillum crustostum	14534	3.2 x 106	0.40 ± 0.24	0.80 ± 0.37	1.00 ± 0.44	1.40 ± 0.24	4.67	1.38^h
		2.4 x 106	0.20 ± 0.20	0.60 ± 0.40	1.00 ± 0.44	1.20 ± 0.37	4.00	0.69^h
		1.6 x 106	0.00 ± 0.00	0.40 ± 0.24	0.60 ± 0.40	1.20 ± 0.20	4.00	0.69^h
		0.8 x 106	0.00 ± 0.00	0.40 ± 0.24	0.80 ± 0.37	1.00 ± 0.44	3.33	0.00^h
		0.4 x 106	0.00 ± 0.00	0.60 ± 0.40	0.80 ± 0.37	1.00 ± 0.31	3.33	0.00^h
Control	-	-	0.00 ± 0.00	0.40 ± 0.24	0.60 ± 0.24	1.00 ± 0.31	3.33	0.00^h

Data are presented as mean value (no. of dead mites) ± SE. a, b Different letters indicate a significant difference according to Duncan’s multiple range tests. Values are significant at p= 0.05 levels, df =129, F-value= 56.39 (after 9 days).

Table (3) Estimates of the LC ₅₀ and LC₉₀ values of R. robini mite treated with entomopathogenic fungi.

Fungi	LC₅₀ value conidia/ml.	LC₉₀ value conidia/ml.	Slop	LT₅₀
Aspergillus terreus 1	1.06 x10⁶	2.44 x10⁷	0.941	6.77
Aspergillus terreus2	3.45 x10⁶	4.5 x10⁷	0.847	8.09
Aspergillus flavus	7.42 x10⁹	6.83 x10¹⁴	0.258	18.16
Myrothecium advena	1.75 x10⁶	2.83 x10⁷	1.060	6.62

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Effectiveness some Egyptian isolates of entomopathogenic fungi against bulb mite (Rhizoglyphus robini - Acaridae)

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Abstract

Figures

Introduction

Materials And Methods

1-Isolation, purification, and identification of entomopathogenic fungi:

2-Identification of entomopathogenic fungi by morphological methods:

4- Mites Culturing:

Result

1-Isolation and identification of entomopathogenic fungi:

Discussion

Declarations

References

Tables

Additional Declarations

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