Ecological Biodiversity Measurement of Seed Mycoflora Contamination of Freshly Harvested in Maize Growing Zone-II

Maize is considered third most important cereal crops in the world. In our study the seed mycoflora of freshly harvested maize of zone-II were isolated by Agar plate method (APM) and Blotter plate method (BPM). A total of 9 genera i.e. Aspergillus flavus, A. niger, Bipolaris maydis, Curvularia lunata, Fusarium verticilioides, Penicillium notatum, P. expensum, Rhizopus stolonifer, and Rhizoctonia solani were isolated by standard Agar plate method and 10 fungal genera, i.e. Alternaria alternata, Aspergillus flavus, A. niger, Bipolaris maydis, Curvularia lunata, Fusarium verticilioides, Macrophomina phaseolina, Penicillium notatum, Rhizopus stolonifer, and Rhizoctonia solani by blotter plate method. On the basis of density, frequency and abundance, Aspergillus flavus, A. niger and Rhizopus stolonifer were found as dominate and taken for detail study. The seed lot of this zone is three categories i.e. Original (OS), Partial discolour (PDS) and Discolour seed (DS). Maximum important value index (IVI), Simpson index of dominance (D), Shannon-Weaver index of diversity (H) and Evenness (E) of Aspergillus flavus OS (86.657%, 0.0834, 0.359, 0.184), PDS (63.827%, 0.0453, 0.329, 0.150) and DS (83.467%, 0.0774, 0.356, 0.183) were contributed. In Blotter plate method, highest density of A. niger OS (5.850), A. flavus PDS (4.500), DS (5.225) were recorded. Maximum frequency showed by A. flavus (100.000%) in all categories. The abundance of A. flavus OS (0.350), PDS (0.342), DS (0.407) were recorded. Relative density maximum recorded in A. niger OS (26.401%), A. flavus PDS (19.268%) and DS (26.381%). Relative frequency (RF) and relative abundance (RA) highest were found in A. flavus OS (24.691%, 35.009%), PDS (23.256%, 34.221%) and DS (23.810%, 40.661%). Maximum IVI, Simpson index of dominance, Shannon-Weaver index of diversity and evenness contributed A. flavus OS (80.912%, 0.0727, 0.353, 0.170), PDS (76.744%, 0.0654, 0.349, 0.159) and DS (90.852 %, 0.0917, 0.362, 0.174). These species are some of the common on the maize during storage and spoil the grains.

Maize (Zea mays L.) is a staple food for approximately 400 million people in the worldwide for processed food and feed 1 .In India, maize ranks fifth in total area and third in total production and productivity.It is susceptible to a numerous fungal species that cause ear and kernel rots including, Aspergillus, Fusarium verticillioides, F. proliferatum, F. subglutinans, Gibberella zeae Penicillium, Macrophomina phaseolina, Diplodia, Nigrospora, Botryosphaeria, Cladosporium, Trichoderma, Rhizoctonia, and Rhizopus 2,3 .There has been continuous increase in the world population then consumption demand of corn to be increase in the demand from poultry and piggery sector used as a feed.In the presence of seed borne pathogens several types of abnormalities occur in the seeds.Such seeds are rejected by seed industries and for agricultural purposes.Since the fact endeavor has been made to study the maize seed mycoflora and their cheaper eco-friendly management.Seed borne mycoflora is one of the major components reducing the maize yield.Mycoflora associated with seeds both internally and externally are responsible for seed major step is to use disease free and certified seed 4,5 .Fungal species are related to corn mostly belong to Apergillus spp Fusarium spp.and Penicillium spp.There are many reports that indicate these fungal species produce dangerous mycotoxin which can be harmful for human health and animals 6,7,8 .Usually, fungal species diversity is one of the most important indices used to evaluation of an ecosystem.A large value of Shannon-Wiener Index (H) has showed a rich ecosystem with high species diversity and low value (H') will have a low species diversity 9,10 .The present study was aimed at ecological biodiversity measurement of Seed mycoflora contamination of freshly harvested in maize growing zone-II.

MATERIAL AND METHODS
The maize growing area in to three zones, i.e. zone-I, (Almora, Kullu, Bilaspur, Daulakauna Kangra and Saharanpur) zone-II (New Delhi, Karnal, Pantnagar and Ludhiana) and zone-III (Varanasi and Begusarai).In this study, four maize seed samples were taken from maize growing Zone-II.The collected seed samples of each maize variety will be critically examined and grouped into three categories with the help of hand lens i.e. original seed (OS), partially discoloured seed (PDS) and discoloured seed (DS).Myco-flora detected on maize seed by Agar plate method-APM 11 and Blotter plate method-BPM 12 .One hundred seeds of each category of different varieties untreated will be place in a plastic Petri plates (90 mm dia.) lined with two layers of blotting papers moistened with distilled water for studying the association of different myco-flora with maize seeds.Ten seeds will be placed in each Petri plates equidistantly (pattern-1-3-6).The Petri plates will be incubated at 25 ± 1 °C for five days and the seeds will be examined regularly for the presence of different fungi.There will be two replications each having 50 seeds.Incubated seeds will be examined visually and under Stereo-zoom microscope for the associated myco-flora.The associated fungi were isolated on PDA for further identification.Same method applied in Agar plate method also.The seed mycoflora were identified with the help of literature [13][14][15][16][17][18][19][20] .
Based on the individuals fungi recorded in the distinct seed samples were analysed for density, frequency, abundance, relative density, relative frequency, relative abundance, importance value index, Simpson index of Dominance, Shannon-Weaver Index of Diversity and evenness.The importance value index of seed sample was determined as the sum of relative frequency, relative density and relative dominance 21  The maximum importance value for any one genus is 300 (100 + 100 + 100).It is useful, as it provides an overall picture of the density, frequency and cover of a genus in relation to community.Simpson's Dominance Index (D) -The Simpson's index (D) is calculated using the following equation 23 : Where 'ni' is the proportion of individuals of the ith species in the community.Simpson's index gives relatively little weight to the rare species and more weight to the common species.It weighs towards the abundance of the most common species.It ranges in value from 0 (low diversity) to a maximum of (1-1/s), where s is the number of species.In nature the value of d ranges between 0 and 1.With this, index 0 represents infinite diversity and 1, no diversity.The bigger the (D) value, the smaller the diversity.Shannon-Wiener Index (H)-This is a widely used method of calculating biotic diversity in aquatic and terrestrial ecosystems and is expressed as SWI 24 : Where, H= index of species diversity s= number of species ni= proportion of total sample belonging to the ith species.
Evenness Index (E) -This is relative distribution of individuals among taxonomic groups within a community and is expressed 25 as: E= H'/logS Where, H' = Shannon -Wiener diversity index, and log S= Natural log of the total number of species (S defined as Species Richness) recorded.

RESULTS AND DISCUSSION
Working seed samples were collected from zone-II (New Delhi, Karnal, Pantnagar and Ludhiana).In this study, four maize seed samples were taken from maize growing Zone II category.
This finding was in line with the works of Mudili et al. [26] showed the diversity of fungal species, including frequency, density, and diversity indices such as Important value index, Shannon-Wiener index (species richness) and Simpson index (diversity of species) in 150 freshly harvested maize samples from southern India.Fusarium was the prevailing genus in Karnataka (42%) and Andhra Pradesh (46%), followed by Aspergillus (32 and 33% respectively).In Tamilnadu, was observed highest Fusarium incidence (75%), followed by Penicillium (13%) and Aspergillus (12%).In Karnataka, Aspergillus flavus and Aspergillus niger were observed with 100% frequency while in Andhra Pradesh, in addition to these two Aspergillus species, Penicillium chrysogenum and Fusarium graminearum also showed 100% frequency.In Tamilnadu, Fusarium verticillioides and F. proliferatum were less frequent and highly dense with IVI values of 52.7 and 59.8 respectively.The species richness diversity index (Shannon index) showed that Andhra Pradesh and Karnataka were highly diversified, with several toxigenic moulds, whereas in Tamilnadu the diversity of fungal species was less.
Fungal infection is affected quality of grain through reduction in germination, increase in fatty acids, discolourization, mustiness and spoilage of the grain.Fungal development in grains is influenced by temperature, humidity and storage period.Several literature displays that a number of fungal genera viz., Aspergillus, Fusarium, Penicillium, Bipolaris maydis, Alternaria, Cephalosporium, Macrophomina, Diplodia, Nigrospora, Botryosphaeria, Cladosporium, Trichoderma, Rhizoctonia and Mucor have been reported from maize seed 2,27,28 .
Tsedaley and Adugna 29 a total of 110 fungi isolates were recovered from three maize variety samples in six treatment combinations which is collected in three maize storage conditions, were harvested during 2013 cropping season.Aspergillus, Fusarium and Penicillium are the most prime fungal genera's attacking maize seed and decreasing seed germination.The highest frequency of Aspregillus spp.(40.4%) at farmer preserved seed with surface disinfected kernels on agar plate were recorded.The highest relative density of Fusarium spp.(51%) was only recorded on agar plate test from the farmer preserved seed without surface disinfected kernels.Without sterilized seeds preserved by farmers were recorded lowest germination percentage (62%).The Aspergillus spp.are the most dominant fungi followed by Fusarium spp.isolated.These fungi are important in producing secondary metabolites, which are carcinogenic to both humans and animals.
El-Shanshoury et al., 33 deal with forty food grains including maize, wheat, rice and peanut seeds were analyzed for fungal contamination.Eight fungal genera belonged to Aspergillus, Penicillium, Fusarium, Mucor, Cladosporium, Trichoderma, Rhizopus and Alternaria were isolated and identified.Total fungal loads as CFU and percentages of fungi in the analyzed samples ranged between 21.7-33.2x103CFU/g and 1.6-36.7%,respectively.Contamination of grains with aflatoxins was in the following order; rice > peanut > wheat > maize.In the cultures of Aspergillus flavus Link isolates, AFB1, AFB2 and AFG1 were detected in 78%, 71%, and 36% of the isolates.
Sreenivasa et al., 30  Mostafa and Kazem, 31 reported that means of incidences Fusarium spp.were the highest (35.2%) followed by species Aspergillus, Penicillium, Rhizopus, Mucor and Alternaria i.e., in per cent 2.9, 1.1, 2.3, 1.4 and 0.2 in that order.Among Fusarium species, F. proliferatum (90.1, 42.6%) had the highest percentages of frequency and the highest incidence in Gorgan.Aspergillus flavus had revealed frequency (2%) and incidence (40.2 %) and the highest level of infection was belonged to Bandare gaz seeds studied.Penicillium spp.were isolated from most samples examined which the highest incidence (2%) was in seeds studied in Kalale.
Niaz and Dawar 4 was used blotter, agar plate and deep freezing methods as recommended by ISTA.In all sample, 70% of the samples were infested with Aspergillus flavus, A. niger, A. wentii and Penicillium spp.Among the three methods used, agar plate method yielded the highest number of fungi as compared to blotter and deep freezing methods.Deep freezing method was the best for the detection of Drechslera spp., Fusarium spp., and Penicillium spp., whereas agar plate method was suitable for the detection of Aspergillus spp., Cladosporium spp., Curvularia spp., and Rhizopus spp.
On the basis of present study Aspergilus flavus and Aspergillus niger were recorded dominant mycoflora.So, the next step is monitoring the mycotoxin production of isolated species.
. Density is calculated by the equation: Frequency (%) is calculated by the equation: Abundance-It is the study of the number of individuals of different species in the community per unit area.It is represented by the equation: Relative density, relative frequency and relative abundance was calculated as: Importance Value Index (IVI)-It was calculated by equation 22 -IVI = Relative frequency + Relative density + Relative dominance,

Table 2 .
Biodiversity analysis of Seed mycoflora in maize by Blotter plate method : Ct=Categories, OS= Original Seed, PDS= Partial Discolour Seed, DS= Discolour Seed, Dn=Density, F= frequency, A= Abundance, RD=Relative Density , RF= Relative frequency, RA= Relative abundance, IVI= Importance value index, D= Simpson index of Dominance, H= Shannon-Weaver Index of Diversity, E= Evenness Note a total of 86 maize samples were analyzed for frequency and relative density of internal mycoflora by direct plating method on PDA and MGA 2.5 agar medium.The most prevalent fungal genera occurring on maize grains were species of Fusarium and Aspergillus.The other genera included Penicillium, Drechslera, Nigrospora, Curvularia, Alternaria, Chaetomium and Phoma.The data revealed the high frequency of Fusarium species (96.5%) and the high relative density of Aspergillus species (41.7%) among the 17 fungal genera recoded.The predominant fungi recorded Fusarium verticillioides, F. anthophilum, F. proliferatum, Aspergillus flavus, A. niger and A. ochraceous, respectively.