Succession of Microfungi on Leaf Litter of Acacia catechu in Datia, Madhya Pradesh, India

The present investigation was conducted to find out the fungal diversity on leaf litter samples collected from Ratangarh forest, Datia. The samples were collected from Jan 2017-June 2017 at monthly intervals. Serial dilution method and PDA media was used for the isolation of fungi. During present investigation, 14 species belonging to 6 different fungal genera have been isolated and identified. Out of these, 2 species belongs to class Zygomycota and 12 species belongs to class Ascomycota and their anamorphs. Species namely Mucor hiemalis, Rhizopus stolonifer, Aspergillus niger, Aspergillus japonicus and Torula sp. were early colonizers while Aspergillus nidulans, Aspergillus flavus, Aspergillus fumigatus, Emericella nidulans, Ascochyta sp., Penicillium chrysogenum, Penicillium aurantiogriseum, Trichoderma reesei, and Trichoderma viride were found as late colonizers over leaf litter of Acacia catechu. In all stages of decomposition, fungi belonging to the Ascomycota were predominant. The present investigation provides valuable information about diversity of leaf litter fungi of tropical forest.


INTRODUCTION
Forest produces a large amount of organic matter in the form of litter which provides unique habitats for fungi. Leaf litter fungi are the major degraders of plant litter which is composed of basically cellulose and lignin and play a major role in carbon and nitrogen cycling in the forest ecosystem 1 . Decomposition of plant litter is a physical and chemical process which is involved in reducing litter to its elemental chemical constituents and as such a major determinant of nutrient cycles 2 . Fungi are the major component of soil microbiota in the forest ecosystems which utilize the organic substrates present in leaf litter as energy and nutrient source by the process of decomposition. In their role as decomposers, leaf litter fungi also sequester carbon and move it to the recalcitrant carbon stores of the soil 3 .
In addition to their vital role in ecosystem services, fungi are of great interest for biotechnological application in food, wine and textiles industries⁴ , ⁵, for energy generation⁶, for plastics degradation⁷, and to produce highly-added value compounds such as biosensors, cosmetic products and organic acids⁸ , ⁹.
For the present study, a tropical deciduous forest ecosystem was chosen in Datia, Madhya Pradesh, India (Fig. 2a)

Study site and sample collection
For sampling purpose, the forest area of Ratangarh in Seonda block is selected. This place is 65 Km away from Datia city and situated in the north eastern part of MP, India (25° 28' to 26° 20' N latitude and 78° 10' to 78° 45' E longitude) ( Fig.  1a & 1b). Datia experiences widespread Indian monsoon climate with an average annual rainfall of 825.93 mm. with average maximum and minimum temperatures of 32.64°C and 18.45°C.
Litters of Acacia catechu family have been collected randomly from research field from January 2017 to June 2017 at monthly intervals. Collected leaves were brought to laboratory in sealed polythene bags within 4 hrs of collection for further study.
Approximately 20 g leaves were enclosed in nylon mesh bag 27 , (with mesh size 2 mm) and placed randomly into 0.61 meters pits ( fig. 3a & 3b) (one bag in each pit) for decomposition by soil fungi. One bag from each pit was randomly removed at a regular interval of time (15,30,45, 60, 75, 90, 120, 150, 180 days) after their incubation in pit. Each bag was placed in a separate paper bag and transported to the laboratory for isolation work.

Isolation of fungi
The isolation of litter fungi was carried out by using serial dilution method 28 . 10 g of decaying leaves were transferred into 250 ml Erlenmeyer flask containing 100 ml distilled water and shaken thoroughly for 15 min on a horizontal mechanical shaker for spore suspension. The suspension was further diluted 10 3 and 10 4 times. One ml of this suspension was inoculated separately into each of five Petri plates containing 15 ml potato dextrose agar nutrient medium i.e. PDA (Potato 200g, Dextrose 20g, agar 15g, pH 5.5, Distilled water 1000 ml) supplemented with streptomycin to avoid bacterial contamination in culture plates. The plates were subsequently incubated at 28 ± 2°C.
Development of fungal colonies was noticed from third day of incubation. The heterogeneous or mixed fungal colonies appeared after 5 days of incubation. Desired colonies were transferred to freshly poured Petri plates containing PDA media in order to obtain pure and mono culture. The pure cultures were then preserved on PDA slants and maintained at 4ºC for further studies.

Identification of fungi
The majority of sporulating isolates were identified on the basis of their cultural, morphological and spores characteristics with the help of standard texts and keys [29][30][31] . Preparation of microscopic slide of the isolated fungi, to aid identification, was done using the pure culture. For the preparation of semi permanent slides of fungal fruiting bodies a portion of mycelium of the representative colony was picked up with the help of a pair of needles and mounted on a clean slide with solution of cotton blue in lactophenol. The slide was examined under Trinocular microscope (Olympus) with 10x & 45x objectives and 10x & 15x eye pieces and the microphotographs of the individual fungal species were taken.

RESULTS AND DISCUSSION
Six fungal genera and fourteen species have been isolated and identified from leaf litter of Acacia catechu over a period of six month of decomposition (Table-1 Fig. 4 (a & b). Shows heterogeneous or mixed fungal colonies  In the decomposition process, the initial colonizers are main utilizers of simple organic compounds (sugars) whereas the later once are capable of exploiting complex organic molecules such as cellulose and lignin 41 . Initial colonizers or pioneers gradually disappear and another inhabitant replaced earlier ones. This is possible because of the availability of different kinds of nutrients in different levels of decomposition. The species of Aspergillus, Penicillium and Trichoderma are capable of using cellulose as a source of carbon for their growth and multiplication. The physiochemical environment, litter quality and the composition of the decomposer community are the three main factors controlling litter decomposition [42][43][44] .

Morphological characterization of leaf litter fungi of Datia forest Mucor hiemalis Wehmer
(J) Colonies white, later grey; reverse pale olivaceous grey both in light and dark; sporangiophore with large sympodial branches originating from a short distance below the previous sporangia, sporangia globose, blackish brown, wall diffluent, leaving a basal collarette; columellae globose or oval, sporangiospores variable in shape, cylindrical-oblong 31 .

Rhizopus stolonifer Ehrenberg
(K) Initially colonies were white then become blackish with spreading stolons, internodes brown, rhizoids branched at internode, unbranched. Sporangiophores were in group of 3-10 which become dark brown at maturity. Sporangia appears black, Spores were irregular round and oval, angular in shape and grey in color 31 .

Aspergillus niger Tiegh
(D) Colonies with abundant mycelium, conidial heads are deep black or brownish black; conidiophores arising directly from the substratum, conidia may be globose, spinulose with black pigment, globose to subglobose, sclerotia produced in some strains, at first cream to buff, later vinaceous buff in age 31 .

Aspergillus japonicus Saito
( B ) C o l o n i e s s p re a d i n g ra p i d l y producing purple brown to black conidial heads; conidiophores arising from the substratum, conidia mostly globose, sometimes subglobose, strongly echinulate, bright colored at first, becoming purplish brown 31 .

Torula sp. Peersoon ex Fries
(C) Colonies small, discrete, olive brown or dark brown or sometimes black in color. Mycelium superficial unbranched or sometime branched but irregularly. Conidia blastic dry, in simple or branched chains which arise from surface of the conidiogenous cells. Cells are cylindrical or spherical may break into with phragmoconidia.

Aspergillus nidulans Fenn and Raper
(F) Colonies are grown at room temperature. In some strains colonies are green and conidiophores light brown, sinuous, smooth, phialides biseriate, conidia globose to subglobose, green in mass 31 .

Aspergillus flavus Link
(A) Colonies growing rapidly, at young stage conidial heads are yellow then become yellowish green and then greyish green at older, conidiophores develop from the substratum, conidia globose to subglobose, echinulate, yellowish green, sometimes elliptical when young 31 .

Aspergillus fumigatus Fresen
(C) Initially colonies are velvety to floccose then become bluish green, conidiophores short, smooth, light green and enlarged into a flask shaped vesicle; conidia globose to subglobose, green in mass, echinulate, sclerotia and cleistothecia absent 31 .

Trichoderma reesei E.G. Simmons
(P) Colonies growing moderately, aerial mycelium appressed to agar surface, cottony aerial mycelium not forming, white with radial lines and scant conidial production after one week; conidiophores forming on aerial mycelium, tufts minute, only primary branches infrequently branched, phialides arising singly before first branch, side branches producing more than one phialide; conidia obovoid to ellipsoid, smooth, and light green 31 .

Trichoderma viride Pers.
(O) Colonies appears hairy due to formation of aerial mycelium, glaucous to dark bluish green; conidiophores may be formed on the aerial or creeping hyphae, main conidiophores producing smaller side branches, ultimately a conifer-like branching system is formed, conidia globose or short obovoid, or broadly ellipsoidal, sometimes with distinct apiculus-like base because of distinct minute roughening on their walls, bluish green to dark green 31 .

Penicillium aurantiogriseum Dierekx
(N) Colonies growing moderately, radially sulcate, moderately deep, surface texture smooth to granular, fascicles seen in young growth at low magnifications; margins usually entire, deep, and white; mycelium white, usually inconspicuous, occasionally dominating the colony appearance in floccose isolates; conidiophores borne singly or in fascicles, mostly from substrate hyphae, with stipes, bearing terminal terverticillate or less commonly biverticillate penicilli; conidia subspheroidal to ellipsoidal, smooth, usually borne in long well defined columns 31 .

CONCLUSIONS
The present investigation provides valuable information about the leaf litter fungal diversity of tropical forest. The fungal communities of the tropical forest were entirely dominated with Ascomycota and co-dominated with Zygomycota. The data presented here indicates that Aspergillus niger, Aspergillus japonicus, Aspergillus flavus, Emericella nidulans, Ascochyta sp., Trichoderma spp. and Penicillium sp. may contribute or be useful in future work in the area of nutrient recycling and may also act as fungal bio-indicators of decomposition in the particular forest area of Datia district.