Studies on Air Borne Fungi of Tea Seed Orchard at Gaya Ganga Tea Estate of Darjeeling District, West Bengal

Air borne microorganisms obtaining tremendous interest as they play a significant role in the plant, animal and human health. Studies on aerobiology can help to recognize the resources, take-off, passive deposition and impaction of these microorganisms on the plant life. So far, no work has been reported on the air spora of the seed orchards of tea garden. This work aimed to observe flowering period and the diversity of air mycoflora prevalent in six biclonal tea seed orchards (TS: 569, TS: 491, TS: 557, TS: 520, TS: 463 and TS: 462). A total five species were recorded. Penicillium sp is found to be dominant over the other recorded species. The maximum population of mycoflora (CFU 1580/M3) was recorded under TS: 569 and the lowest (CFU 180/M3) under TS: 463. Air mycoflora fluctuated under TS: 491, TS: 557, TS: 520 and TS: 462 and were CFU/M3 740, 540, 340 and 380 respectively.


Introduction
Tea Camellia sinensis (L.) Kuntze is an out-breeding plantation crop, and as such, cross-pollination is essential to produce seeds and to get superior seeds from better hybrid progenies. 1Tea plants grown nearer to the equator flower throughout the year however, in north-east India, flowering of tea plants starts in October with a peak in November and December.It takes 120-150 days from the appearance of flower buds to their full anthesis. 2 present, the seed yields in tea seed orchards in Assam are low, ranging from 6 to 12.9 percent, 3 which may be due to increasing pollution levels as pollutants are known to affect pollen germination. 4n tea seed orchards, flowers are openly pollinated by long-distance bees or wasps.The role of ants and unidentified black weevils, which are seen inside flowers very often, probably contributes greatly towards self-pollination. 3Pollen germination is affected by offsite application of agro pesticides, 5 which may be attributed to the low yield of seeds.Tea flowers are known to contain similar healthpromoting components and nutraceuticals as tea, 6,7 and local people relish tea flowers as a unique source of nutrition supplements.
Currently, tea plants are propagated by two means: seeds and clones.The demand for seeds in the Indian tea industry is ever-increasing due to the adoption of an uprooting and replanting program. 8ocklai Tea Research Institute, Jorhat, carried out intensive work on tea selection and conventional breeding in the past and released 14 biclonal seed stocks (TS:378, TS:379, TS:387, TS:449, TS:450, TS:462, TS:463, TS:464, TS:491, TS:506, TS:520, TS:557, TS:569, and TS:589).These seed stocks with different parental combinations are supplied to certified seed growers for establishing seed bars or seed orchards in different regions (Upper Assam, North Bank, Terai, Dooars, Darjeeling, and Tripura) of North East India.9A seed bari in Terai is located at Gaya Ganga T.E. and was established with 13 biclonal seed stocks to fulfil the demands of seeds for the region, e.g., TS:378 (14.5.35 * 14.6.28), TS:379 (14.5.35 * 14.12.16),TS:397 (TV1 * 9. Mature tea seeds, after pollination, drop underneath the tea seed plants and, as such, get contaminated with different species of fungi from the soil.Besides airborne mycoflora, they remain associated with leaves, growing capsules, and the surface of seeds.Fungi like Fusarium, Alternaria, Aspergillus, Cladosporium and Penicillium, colonize seed surfaces and are known to produce mycotoxin.Fungal deterioration of tea seeds caused by airborne mycoflora is a significant constraint for seed growers, and fungal mycotoxin-producing activities are likely to increase due to climate change and global warming. 10r pollution causes annual mortality of 9 million people globally and is a growing problem and pose a significant environmental and public health challenges in the face of climate change particularly in developing countries. 11Rapid industrialization, urbanization, destruction of forests for infrastructure, and construction of urban housing have caused emissions of GHGs like CO 2 , NO 2 , and CH 4 that have influenced the air quality index.WHO has recently revised air quality guidelines to include stricter limits. 12Air is composed of various types of gases in different proportion e.g., carbon monoxide (CO), Nitrous oxide (NO), Sulphur dioxide (SO 2 ), formaldehyde, polycyclic organic matter in the form of Benzopyrene suspended as aerosol.Exposure to airborne particulate matter (PM 10 , PM 2.5 ), pollutants containing fungi, pollen grains, bacteria, actinomycetes, algae, etc. and their secondary metabolites can result in respiratory disorders and other adverse health effects such as allergy, infection, hypersensitivity pneumonitis, and toxic reactions. 13,14In India death due to air pollution reaches as high as 1.6 million annually including 28,799 premature deaths. 15st scientific literature shows the negative impact of microbes and is over emphasized at times without critical evaluation.Microbes perform various ecosystem services, including carbon cycle, decomposition of debris, soil health, occupying the rhizosphere zone and contributing to plant health.Airborne fungi influence plants, animals, humans, and some microbes remain intimately associated as endophytes or gut flora and produce metabolites. 16,17Bioactive compounds produced by fungi comprise over 8600 compounds, accounting for 38% of all microbial sources. 18These microbes in humans, animals, and plants influence the hosts' physiology and immune system positively.Microbes are transported vertically to the next generation through seeds and benefit the host's offspring.There is ample evidence of the continuous exchange of microbes among humans, animals, plants, and the environment.However, the use of antifungal agents and antibiotics can inhibit the growth and development of microbes and give rise to antimicrobial resistance.
Aerobiology in India was studied by Cunningham in1889 at Calcutta, and Gregory in 1967 published a book entitled "Microbiology of the Air".Later, a comprehensive review of Indian aerobiology was compiled. 19The Indian Aerobiological Society was established under the presidency of the late Prof. Sunirmal Chanda in 1980.Air sampling was carried out by a portable Microbial sampler, the MicroBio MB1, manufactured and supplied by Cantium Scientific Limited, Dartford Kent, UK.The sampler has an air flow rate of 10-100 l/min.The sampler collects airborne organisms by drawing a stream of air at a constant flow rate through a series of small holes in a metal head.Viable spores, conidia, and hyphal filament particles suspended in an air stream impinge on the surface of a sterile culture medium in a contact plate or Petri dish.
The sampler was placed about 2 feet above ground level beneath tea seed plants between 10 and 11 a.m. on December 25, 2022.A triplicate disposable sterile Petri plate (8cm dia.) containing Rose Bengal Chloramphenicol agar was used.Plates were sealed after exposure, incubated at 30°C in the laboratory for 72 hours and counted after the appearance of fungal colonies.
The colonies of fungi were examined under low and high magnification using light microscope and later identified with the help of available literature. 20,21,22antitative compositions of mycoflora were determined by counting the number of total colonies and percent occurrence of individual taxa was obtained as per following formula given by, 23 with slight modification.

% occurrence of individual species
Colony forming unit per cubic meter was calculated as per following formulae given by, 23 with slight modification.
Where Nc is the number of colonies and Vs is the sampled volume in litres.

Results and Discussion
It was observed that bud initiation was seen during the 4 th week of September, while the peak of flowering was from the 2nd to the 4th week of November in 2022.Flowering of plants slowly declined from the second week of December to trace or no flowering by the end of February.Bee activity was observed in October and long-distance bee, hornets were seen visiting tea seed plants during flowering.The flower, comprising petals and androecium attached to it, drops off the ground below within 48 hours of pollination.The flowers are collected by locals and workers for consumption as nutritious food.
It was evident from the Table 2 that    Qualitatively, a total of three Penicillium species, one Aspergillus niger and one Cladosporium species were recorded.The dominance of Penicillium spp.over other species was observed during the period of study.The high population of Penicillium colonies (P.chrysogenum and P. malochi) may be attributed to increased colonization of flowers that drop on the ground after pollination.
The deep orange colour produced by Penicillium was found to be closely related to monoverticillate P. mallochi.Monoverticillate Penicillium is found to be potent producers of red or orange-red pigment. 24he genus Penicillium was introduced by Link in 1809, probably indicating similarity with brush, and he described three species: P. candidum, P. glaucum, and P. expansum.The taxonomy of Penicillium is highly complex due to the introduction of one fungus with one name by the International Botanical Nomenclature Committee in Melbourne, 2012.The genus Penicillium currently contains 354 accepted species. 25The genus Penicillium has profound agricultural, 26,27 environmental, 27 food, and medical importance. 28,29The first ever antibiotic penicillin was obtained from P. notatum and P. chrysogenum, which were discovered by A. Flemming in 1943.Many of the species of Penicilium produce mycotoxins, e.g., patulin, citrinin, rubratoxin, cyclopiazonic acid, and ochratoxin, which are produced by P. patulum, P. citrinum, P. rubrum, P.cyclopium and P. verrucosum, respectively.Species of Penicillium are also found to produce pigments that are currently used commercially as food colouring. 30,31eep red pigment is produced by P. cyclopium and P. viridicum, 27 while orange pigment is produced by P. purpurogenum and P. marhneffi.During the study of diurnal variation of air mycoflora over a tea plantation in plains of Darjeeling district, observed the dominance of Cladosporium (71% of the total) over other air mycoflora. 32Aspergillus niger was dominant over the standing tea. 33Aspergillus also produces different types of toxins, such as aflatoxins (A.flavus, A. parasiticus, and A. nomius) and ochratoxins (A.carbonarious and A. ochraceous).Besides, species of Aspergillus are widely used in different industrial applications, including the production of foods, drinks, organic acids (citric acid), and a large variety of enzymes (A.niger, A. aculeatus and A. oryzae). 34,35Lovastatin, a cholesterol-lowering drug, is produced by A. terreus. 36Research on Aspergillus occupies one of the priority areas in global academic, R & D and industrial institutes.In food industry A. niger has been accorded GRAS (Generally Recognized As safe) status by the US FDA. 35Species of Aspergillus are cosmopolitan and occur in different ecosystems in a wide range of environmental and climatic zones. 37,38hey are capable of colonizing a wide variety of substrates differing in chemical composition. 39They are found throughout the world in soil, 40,41,42,43 saltproducing areas, 44,45 agroecosystems, 46,47,48,49 polar environment, 50 living plants, animals, and lichens, 51 stones, water-related areas, 52 fossil specimens, 53,54 and human allergy and respiratory ailment. 55,56. niger produces a range of secondary metabolites and mycotoxins and has been comprehensively reviewed.A. niger has also been explored as a biocontrol agent for Fusarium wilt in tomatoes. 57adosporium sp were observed under Tea seed orchards TS:569, TS:557, TS:520, and TS:463 with 300, 20, 180, and 20 cfu/M 3 , respectively.In tea orchards, more than one species of Cladosporium spp. was found to colonize the anther and petals of flowers, which drop off after pollination, and probably serve as the primary source besides the phylloplane environment.It has been stated that phylloplane mycoflora serve as a major source of airborne fungi. 58In the tea ecosystem, leaves serve as habitat for the growth and survival of Cladosporium, the dominant species on the ventral surface. 59e fungus Cladosporium was described by H.F. Link in 1816 with C. herbarum as the type species.The genus Cladosporium sp now has an estimated 772 species. 60adosporium species are cosmopolitan in distribution and commonly encountered on plant, fungal and other debris, compost, decayed flower petals, soil, food materials, wet walls, wall paintings, textiles, and other organic matters, or colonize as secondary invaders leaf lesions caused by plant pathogenic fungi. 61,62Some species are hyperparasites, 63 endophytes, 64,65 and components of phylloplane fungi. 66,67,68,69,70Some species cause human respiratory ailments.

Conclusion
The flowering time of tea plants in foothills of Darjeeling district starts at the end of September and November is the peak flowering time.Tea flowers serve as reliable sources of nutrition supplements for humans.The maximum population of mycoflora (CFU 1580/M 3 ) was recorded under TS 569 and the lowest (CFU 180/M 3 ) under TS 463.Air mycoflora fluctuated under TS: 491, TS: 557, TS: 520 and TS: 462 and were CFU/M3, 740, 540, 340 and 380 respectively.Red-pigmented species i.e., Penicillium malochi found to be dominant over Aspergillus niger and Cladosporium sp.
The low diversity of mycoflora observed in the present study may be attributed to the low temperatures prevailing during the period of sampling.The normal path of air flow under tea orchards gets altered due to the growth of tea plants to a height of 3-4 meters or more.A possible reason for the low incidence may be attributed to the leaf surfaces of plants, which might act as traps for airborne mycoflora as well as dust, which reduce the load as compared to open air.This aspect remains a research gap.There is a need to carry out seasonal variation of air mycoflora in tea seed orchards.Future studies aim at exploring the role of these mycoflora on seed health and seed output.

Acknowledgement
Authors are grateful to the Management of Gaya Ganga T.E. for permission to visit the Estate and carry out the work.Authors are also grateful to Dr.

Sukumar Debnath for providing Portable Microbial
Air Sampler for the present study and valuable suggestions in planning the entire work programme.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Fungal
foliar plant pathogens of tea include Pestalotiopsis theae, Colletotrichum camelliae, Exobasidium vexans, Fusarium solani, Fusarium oxysporum, Cephaleuros parasiticus (algal disease), Phomosis theae, Poria hypobrunnea, etc. Spores are released within the plant surface in the boundary layer, and some spores escape the boundary layer to enter the general air spore.Spores that have an active discharge mechanism can escape the laminar flow effect of the boundary layer and travel a long distance.Exobasidium vexans, which causes blister blight disease in tea, has active discharge mechanism, and therefore basidiospores spread far and wide.On the other hand, spores of Pestalotiopsis, Fusarium and Colletotrichum remain aggregated as mucilaginous masses and spread primarily by rain splash.The aim of the study is to investigate the period of flowering and diversity of air mycoflora prevalent in six biclonal tea seed orchards (TS:569, TS:491, TS:557, TS:520, TS:463, TS:462) in Gaya Ganga Tea Estate.Materials and Methods Aero mycological study was carried out at seed orchards of Gaya Ganga T.E. of Darjeeling District of West Bengal in between of Latitude 26.6101° N. and Longitude 88.3037° E.).Observation on flowering time of seed baries was recorded during 2022 -2023.