Isolation and diagnosis of fungi associated with beekeepers Apis melifera and its effects on some plant pathogenic fungi

Fungi are real-nucleus organisms that are widely present in the environment, some of which are large, such as truffles and mushrooms, and others are small in size, and others are seen only by microscopes such as yeasts, fungi offer many benefits to modern society through their use in the pharmaceutical, beverage and food industries. The study was based on the isolation and diagnosis of fungi associated with beekeepers from different bees from different areas of Samarra district and the isolation lasted for 12 months. The results showed that there is a difference in the number and type of fungal insulation by the different parts of the insect's body on the one hand and the different chapters of the year on the other. Thirty-one fungal isolations from the body parts of the beekeepers' insect diagnosed microscopic isolations into 19 fungal races. Anti-all fungal isolation tests (31 mushrooms) with 5 nurses were conducted: Alternaria solani, Fusarium oxysporum, Fusarium solanio Sclerotinia sclerotiorum and Pythium aphanidermatum using the method of double implantation on the steel medium. This study aimed to isolate and diagnosing fungi accompanying the different parts of the beekeeper's body in Samarra and to assess the efficiency of isolated fungi in resisting some fungal pathogens .


Introduction
Fungi are real-nucleus organisms that are widely present in the environment, some of which are large, such as truffles and mushrooms, and others are small, and others are seen only by microscopes such as yeasts, fungi offer many benefits to modern society through their use in the pharmaceutical, beverage and food industries. On the other hand, it is one of the most important pathogens for humans, animals, and plants with the potential to cause devastating deaths and economic losses. Similarly, fungi also have a negative impact on agriculture, posing a serious threat to our food supplies. Due to the high incidence of fungal diseases affecting plants coupled with increased resistance of these fungi to pesticides at an alarming rate, the frequent and indiscriminate use of pesticides has caused many environmental problems, including pollution of water, soil, and food, as well as contamination of agricultural products and the elimination of beneficial microbiology in the soil, the development of bio-control programmes for plant diseases, has become an urgent necessity to reduce the negative effects of pesticide use to include the use of bio-resistance by introducing bio-organisms. Anti-growth organisms of fungi, bacteria, yeasts, and their metabolic products (Tracy et al., 2018(. Fungi also coexist with insects or form internal plant fungi and root colonies due to their presence in the soil, anti-plant diseases, and plant growth catalysts provide these newly understood features possibilities for using fungi in multiple roles where some fungal species can simultaneously suppress plant pathogens and parasitic nematodes as well as promote plant growth (Lacey et al, 2015). Fungi and insects have coexisted over the past 400 million years, and their interactions have developed in different ways. (Mannino et al., 2019(. The coexistence of many fungi with insects may be intrusive or intrusive, and intrusion may be internally Endoparasites or externally Ectoparasites, and all insect ranks are at risk of fungal infection (Scholte et al., 2004). Fungi are naturally present on honeybee insects, most of which cannot Original Research Article

Science Archives (ISSN:2582-6697)
Journal homepage:www.sciencearchives.org journal homepage: www.elsevier.com/locate/enmm penetrate the blood and injure the insect or grow in its cells, but the injury can occur as a result of ingestion of fungal spores and germination within the insect (Sharif, 2012).
Statmets and others have indicated that honeybees may gain health benefits from fungi and their antimicrobial compounds. Western honeybee Apis mellifera is an important component of crop production and food diversity around the world, with A. mellifera and other Apis also playing a key role in the environmental stability of wild plants within endemic areas of Europe, Africa and Asia. from 75% of honeybee colonies. Bees are the main pollinators of insects for plants. Many seed and fruit farmers in the United States keep large numbers of bees or rent them from bees, spending several million dollars a year on pollination services (Tauber et al., 2019). Honeybees are important pollinators for commercial blueberries in the southeastern United States, and blueberry producers often rely on bees to pollinate for high fruit production, as indicated by the results of a 3-year field study conducted by Dedej et al., (2004) to test the hypothesis that the use of beehives equipped with dispensers containing bio-control product Serenade, a commercial combination of Bacillus subtilis bacteria that showed activity against floral infection In laboratory trials, the results of the study showed that using honeybees as pollinators to berries can reduce the incidence of mummy berries and that this is a wise practice that improves the benefits of pollination through the use of bees as a vital vector for the element used in bio-continence .
Because of the importance and spread of fungal diseases on field crops in the country in general and Salaheddine province in particular and the absence of a prior study to assess the efficiency of fungi associated with bee insect factors as vital control factors against plant nurses, this study was conducted to isolating and diagnosing fungi accompanying the different parts of the beekeeper's body in Samarra and to assess the efficiency of isolated fungi in resisting some fungal pathogens.

Sample collection
Samples were collected from various bees in Samarra district for a period of (December 12, 2019-December 12, 2020) for the purpose of isolating fungi associated with bee insect workers, diagnosing them both superficially and figuratively, and assessing their efficiency in inhibiting a number of plantpathogenic fungi. The observations, which include the date of the sample and the name of the area from which it was collected, were recorded, and transferred to the laboratory for the purpose of isolation, purification, and diagnosis.

Insulation of insect-accompanying fungi
The samples were transferred to the laboratory and each insect was placed in a petri dish containing distilled water sterile for the purpose of washing and for one minute and then transferred to other dishes containing sodium hypochlorite solution NaOCL at a concentration of 1% and for 30 seconds to dispose of fungi on the outside surface and then transferred to other dishes containing sterilized distilled water to wash them from the residue of sodium hypochlorate solution and then placed on sterile filtration sheets for drying. Each insect was then cut into five parts: head, chest, abdomen, legs, wings, with a sterile scalpel, then the parts were moved by sterile tongs and planted in petri container dishes on the transplant circles (PDA, SDA, ME A) And in fact 3 similar pieces in each petri dish with a diameter of 9 cm, and wrote the required notes on each dish and includes the number of the part and its name, the date of insulation and the name of the medium used and then hugged the dishes at a temperature of 25 ± 2 C° for 7 days After the end of the incubation period and the growth of the fungus accompanying the insect, the insulation was purified by taking part of each developing fungal colony by a sterile lobe and transferred to a petri dish containing the PDA implant medium and incubated at a temperature of 25 ± 2 C° for 7 days (Chauhan and Jindal, 2020) After the growth of innate insulation was encoded with semantic keys, the developing insulation was given numbers, the first number indicating the part of the insect from which it was taken, the second number indicates the refined part and the third letter indicates the name of the isolated medium, and after the second incubation period the isolations were divided outwardly by the color and strength of the innate colony and divided on this basis into isolation .

Direct antibody test on the solid medium to assess the efficiency of fungi associated with beekeepers against plant pathogenic fungi
We tested the anti-fungus efficiency associated with isolated beekeepers with dual Culture Technique nurses with petri dishes containing PDA implant medium and to revise the center of each half dish with a disc (0.5 cm diameter) from nurses' colonies Each of the 7-day-old mushroom farm was taken by a sterile cork piercer and at the same time to be vaccinated in the middle of the other half with a tablet taken by isolated fungus colonies, and by three repeaters per isolation and incubated dishes at a temperature of 25±2 After 7 days, the contrast was estimated according to the five-year standardization scale prepared by (Bell et al., 1982), as follows: • The growth of the fungus accompanying the insect covers the entire area of the dish without allowing the insulation of pathogenic fungi to grow . • The growth of the insect-accompanying mushrooms covers three quarters of the dish, and the growth of pathogenic mushrooms covers the remaining quarter . • The growth of the insect-accompanying mushrooms covers half the area of the dish and the pathogen's growth covers the other half with no separation zone between the two colonies. • The growth of the mushrooms accompanying the insect covers a quarter of the area of the dish while the nurse's growths cover three quarters of the dish . • The insect-accompanying mushrooms do not grow, and the pathogen covers the entire area of the dish .

Antibody test using fungal spray to assess the effectiveness of bee-accompanying fungi against nurses
Malt Extract Broth was attended and distributed in 250 ml glass rotors, at a rate of 250 ml/dork, closed the glass rotors with tampons and sterilized with the electric bumper and after the sterilization process was completed left the rotor at a temperature The room to cool and then add the antibiotic chloramfinicol by (62.5g/250ml) per dork and vaccinate the rotor with 0.5 cm diameter tablets from each isolation of the fungus accompanying the bees, I hugged the rotor at 25+2 m temperature for 28 days taking into account the swing of the rotor every 2-3 days for the purpose of distributing innate growth, the filters were filtered after the end of the incubation period by filter paper (whatman No. 1) for the purpose of separating condescends and fungal threads from the fungal spray, then placing the spray in tubes discarded using centrifuge at 5000 rpm for 3 minutes to get rid of the micro impurities of the center and mushroom growths and to ensure the purity of the filter refilling process was restored Using a 0.22 μm sterile Millipore membrane filter, the spray was distributed in sealed tubes and kept in the refrigerator until use . The results were statistically analyzed by the application of Minitab statistical program, Ver.Ver17. Use the ANOVA test and compare the calculation averages of different transactions with the Dunkin' multi-border test with a probability level of 0.05 and 0.01. (Al-Rawie, 2002(.

Results
The results of isolation and diagnosis showed the presence of many fungi associated with bee insect factors where 31 fungal species dating back to 19 species were diagnosed, including two yeasts isolated from the body parts of bee insect workers, as in table (1).
Exactly 31 was isolated, diagnosed fungal species belonging to 19 species, from different body parts of the bee insect (head, chest, abdomen, wings, and legs) and the results showed that isolated fungi belong to six people and the vast majority of them belonged to the missing fungi Deuteromycota and by 83.87% of the number of species and 73.68% of the number of races followed by the insethical fungi Zygomycota, from which two species were isolated, Rhizopus stolonifern and recomosus Mucor, accounting for 6.45% of the number of species and 10.52% of the number of races followed by the egg fungus Oomycota where isolated from them one species due to the sex Pythium and cystic fungi Ascomycota isolated from them one race belongs to yeast Saccharom Basidomycota, represented by the rhodotorula yeast genus, appeared in equal proportions, which are 3.22% of the number of races and 5.26 of the number of species. The results of the insulation of the fungi included in the figure (1) show that the highest rate of innate growth was of the legs at 80.64%, which may be due to the friction of the legs with wherever the insect stands as well as having legs in it a basket collecting pollen that may carry some of the fungal spores and each insect has 3 pairs of legs and the least of which was the fungi isolated from the abdominal area and head with 25.80% and 54.83 %respectively.

Ulocladium chartarum (Preuss) Simmons 31
The results showed very clear differences in fungi associated with bee insect agents in their effect on the growth of A. solani pathogenic mushrooms, with A. niger A. fumigatus and T. hazarianum showing the highest anti-pathogenic mushrooms A. solani at an anti-1 degree. On the double farm with a inhibition rate of 100% and showed a very clear superiority over the rest of the other isolations as in the figure (2), followed by the isolation of mushrooms A. parasiticus with an opposite score of 1.5 and a inhibition rate of 87.5%.
For isolations A. flavus, H. toruloide, M. recomosus and P. aphanidermatum, the degree of contrast was 2 and the inhibition rate was 75%, followed by insulation, A. tamarii, C. lunatao, E. nigrum and R. stolonifern with an opposite score of 2.5% and an inhibition rate of 62.5%. Insulations A. alternata, A. citri, A. ocraceus, Aspergillus sp, B. spicifera, C. cladosporioides, C. herbarumo, F. oxysporum and F. Solani, P. chrysogeniumo, P. corylophilum, penicillium sp, Sclerotium sp, T. radicuso, U. chartarum showed a 3% antiism score and an inhibition rate of 50%. Rhodotorula sp and S. sclerotiorum in both were antithesis at 3.5% and inhibition rate of 37.5%, while yeast Saccharomyces sp gave the lowest contrast score of 4 and an inhibition rate of 25%. As shown in figure (2).

Discussion
Our study found that missing fungi are the most present on insect bodies and may be due to the widespread spread of these fungi for their ability to adapt to inadequate environmental conditions and their ability to grow in various natural and industrial agricultural circles alike due to their limited food needs as well as their production of large numbers of colonies (Seifert et al., 2008) .
This study is consistent with what Barbosa et al., (2018) found that isolating 21 species of fungi, including 6 penicillium species and 4 Talaromyces species of bee nests, pollen, and honey, consistent with Elsevier and Prest results (1972) who isolated 38 fungal culture from the intestinal contents of bee workers, including four species belonging to the Penicillium genus, two species of the Aspergillus genus and two species of Cladosporium, and this result surpassed what Gilliam and Morton (1974) found, who isolated 18 species. Some kind of fungus from the intestines of bees, it is in line with the Study of Al-Jumaili (2017), which isolated 28 species of fungi from mosquitoes dating back to 14 species, the majority of which were due to missing fungi. It is consistent with Ghanmi (2016), isolating 39 species of fungi from mosquito larvae Cx. quinquefasciatu and agreeing with the results of Kassiri et al., (2015), who isolated 28 species of fungi from the domestic fly, the most important of which are Aspergillus spp, Penicillium spp, and Rhodotorula sp.
The highest rate of innate growth was from the legs at 80.64%, which may be due to the friction of the legs with any place where the insect stands, as well as having legs in which the pollen collection basket, which may carry some fungal spores, and each insect has 3 pairs of legs and the lowest percentage was fungi isolated from the abdominal area where it reached 25.80%. Fungi isolated from the head area appeared at 54.83% and we can explain this because the head contains the mouth and is in direct contact with flowers to absorb nectar from it and flowers may be infected with some fungi and move to the parts of the mouth and the rest of the head, followed by fungi isolated from Wings are 48.38%, followed by fungi isolated from the chest area and 32.25%, and these results are consistent with some studies that have indicated that fungi are able to stick to insect skin and penetrate the skin through condescending. With insect skin through unspecified interactions (Pedrini, 2017). Batta (2018) also noted that fungi can be used oral and respiratory as an alternative to penetrating the skin of the host insect, which increases the possibility of fungi to coexist with insects.
The results showed very clear differences in fungi associated with bee insect agents in their effect on the growth of A. solani pathogenic mushrooms, as the isolations A. niger, A. fumigatus and T. hazarianum showed the highest antipathogenic effect against A. solani pathogenic mushrooms with an opposite score of 1 on the double farm and in proportions. A 100% inhibition that showed very clear superiority over the rest of the other isolations, as in figure (2). According to Bell et al., (1982), T. hazarianum has a direct intrusion mechanism on the fungal yarn of pathogenic mushrooms by circumventing its filaments and analyzing its walls by releasing its analyzed enzymes, as well as the mechanism of competition for space and food and its rapid growth, as well as its production of antibiotics that negatively affect the growth of pathogenic fungi and limit their reproduction (Zewain and Hamad, 2020). Panaccione and Coyle (2005) stated that mushrooms A. fumigatus produce secondary metabolism substances such as 1,2,3, trimethoxy benzene, and dimethoxy phenol-2.5 which is the reason for its high ability to contrast with some types of fungi and pathological bacteria .

Conclusion
Isolating insect-accompanying fungi from different parts of the body individually and on several transplant circles and in different months of the year has an important role to play in obtaining the largest number of fungi . Bees have been found to be one of the most important factors in the transfer of pathogenic fungi from one plant to another, as well as bioconfictation factors. The presence of the type Aspergillus niger in all parts of the insect's body and in all months of the year .

Consent for publication
The author declares that the work has consent for publication.

Funding support
The author declares that they have no funding support for this study. Al-Ghanimi, Rahman, A.A., (2016). Compare the efficiency of some fungi and their metabolism products in the fight against Culex quinquefasciatus mosquitoes. Master's Thesis / Faculty of Science / University of Qadisiyah. 123 pages. Al-Jumaily, Sarah Nayer Abdul Amir (2017). Assess the efficiency of some fungi associated with Culex quinquefaciatus mosquitoes in combating them in their lifetime. Master's Thesis / Faculty of Science / Qadisiyah University, pages 101.