Shrvan Kumar, Asha Sinha* and Shakshi Singh

Mycology and Plant Pathology, IAS, Banaras Hindu University, Varanasi-221 005, India.

ABSTRACT

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.

Keywords: Aspergillus flavus, A. niger, Zea mays, Simpson index of dominance and Shannon-Weaver index of diversity.

INTRODUCTION

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].

Density is calculated by the equation:

Density = Total number of individuals of a species in all Petri plate
Total number of Petri plate studied

Frequency (%) is calculated by the equation:

Frequency (%) = Number of Petri plate in which the species occurred X 100
Total number of Petri plate studied

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:

Abundance = Total number of individuals of a species in all Petri plate
Total number of Petri plate in which the species occurred

Relative density, relative frequency and relative abundance was calculated as:

Relative density = Number of individuals of a species X 100
Number of Petri plate studied
Relative frequency = Number of occurrence of the species X 100
Number of occurrence of all the species
Relative abundance = Total basal area of the species X 100
Total Petri plate of all the species

Importance Value Index (IVI)- It was calculated by equation [22]-

IVI = Relative frequency + Relative density + Relative dominance,

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.

A total of 9 genera were recorded within three seed categories through Agar plate me00thod. Association of Aspergillus flavus, A. niger, Bipolaris maydis, Curvularia lunata, Fusarium verticilioides, Penicillium notatum, P. expensum, Rhizopus stolonifer, and Rhizoctonia solani were observed (Table-1).  Maize mycoflora was presented with 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 (Table-2).

In Agar plate method, Highest density and relative density of A. niger OS (5.250, 26.960), and DS (5.267, 25.303) were recorded.

Density, frequency, abundance of A. flavus OS (4.875, 100.00, 0.356), PDS (4.800, 75.00, 0.258) and DS (4.925, 100.00, 0.348) were observed. Relative density, highest frequency, abundance by A. flavus OS (25.034, 25.974, 35.649), PDS (19.948, 18.072, 25.806) and DS (23.661, 25.000, 34.806) were recorded. Highest Important value index (IVI), Simpson index of dominance (D), Shannon-Weaver index of diversity (H) and evenness (E) of A. 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.

Diversity of myco-flora in the study calculated using the Shannon-Weiner diversity index (H’) showed values range OS (0.359-0.086), PDS (0.329-0.051) and DS (0.356-0.126). The values for Simpson index of dominance ranges were OS (0.0834-0.0005), PDS (0.0453-0.0001) and DS (0.0774-0.0015). Pielou’s evenness index of myco-flora in OS, PDS and DS samples showed value ranges of 0.184-0.044, 0.150-0.023 and 0.183-0.065, respectively (Table -1).

In Blotter plate mathod, Highest density were recorded A. niger OS (5.850), A. flavus PDS (4.500), DS (5.225). Maximum frequency and abundance values A. flavus OS (100.000%, 0.350), PDS (100.000%, 0.342) and DS (100.000%, 0.407) were showed. Relative density maximum recorded in A. niger OS (26.401%), A. flavus  PDS (19.268%) and DS (26.381%). Relative frequency and relative abundance highest in A. flavus OS (24.691%, 35.009), PDS (23.256%, 34.221) and DS (23.810, 40.661%) were intended. 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), respectively (Table-2).

Diversity of myco-flora in the study considered using the Shannon-Weiner diversity index (H) showed values range OS (0.353-0.054), PDS (0.349-0.112) and DS (0.362-0.122). The values for Simpson index of dominance ranges were OS (0.0727-0.0002), PDS (0.0654-0.0011) and DS (0.0917-0.0014). Pielou’s evenness index of myco-flora in OS, PDS and DS samples showed value ranges of 0.170-0.026, 0.159-0.051 and 0.174-0.059, respectively.

 

Table 1: Biodiversity analysis of Seed mycoflora in maize by Agar plate method
Zone II
Ct Species Dn F

(In %)

Ab RD

(In %)

RF

(In %)

RA

(In %)

IVI

(In %)

D=Pi*Pi H=-{(pi) × ln(pi)} E={H/ln(S)}
OS Aspergillus flavus 4.875 100.000 0.356 25.034 25.974 35.649 86.657 0.0834 0.359 0.184
Aspergillus niger 5.250 70.000 0.269 26.960 18.182 26.874 72.015 0.0576 0.343 0.176
Rhizopus stolonifer 4.367 75.000 0.239 22.424 19.481 23.949 65.853 0.0482 0.333 0.171
Penicillium expensum 1.300 25.000 0.024 6.676 6.494 2.377 15.546 0.0027 0.153 0.079
Fusarium verticilioides 0.864 55.000 0.035 4.435 14.286 3.473 22.194 0.0055 0.193 0.099
Penicillium notatum 1.818 55.000 0.073 9.337 14.286 7.313 30.935 0.0106 0.234 0.120
Bipolaris maydis 1.000 5.000 0.004 5.135 1.299 0.366 6.800 0.0005 0.086 0.044
PDS Aspergillus flavus 4.800 75.000 0.258 19.948 18.072 25.806 63.827 0.0453 0.329 0.150
Aspergillus niger 4.067 75.000 0.219 16.901 18.072 21.864 56.837 0.0359 0.315 0.143
Rhizopus stolonifer 2.300 25.000 0.041 9.559 6.024 4.122 19.705 0.0043 0.179 0.081
Penicillium expensum 0.917 60.000 0.039 3.810 14.458 3.943 22.210 0.0055 0.193 0.088
Fusarium verticilioides 1.654 65.000 0.077 6.873 15.663 7.706 30.242 0.0102 0.231 0.105
Penicillium notatum 0.500 5.000 0.002 2.078 1.205 0.179 3.462 0.0001 0.051 0.023
Rhizoctonia solani 2.000 5.000 0.007 8.312 1.205 0.717 10.233 0.0012 0.115 0.052
Bipolaris maydis 3.000 5.000 0.011 12.468 1.205 1.075 14.748 0.0024 0.148 0.067
DS Aspergillus flavus 4.925 100.000 0.348 23.661 25.000 34.806 83.467 0.0774 0.356 0.183
Aspergillus niger 5.267 75.000 0.279 25.303 18.750 27.915 71.968 0.0575 0.342 0.176
Rhizopus stolonifer 4.300 75.000 0.228 20.658 18.750 22.792 62.200 0.0430 0.326 0.168
Penicillium expensum 1.900 25.000 0.034 9.128 6.250 3.357 18.735 0.0039 0.173 0.089
Fusarium verticilioides 1.000 55.000 0.039 4.804 13.750 3.887 22.441 0.0056 0.194 0.100
Penicillium notatum 1.423 65.000 0.065 6.837 16.250 6.537 29.624 0.0098 0.229 0.117
Curvularia lunata 2.000 5.000 0.007 9.609 1.250 0.707 11.565 0.0015 0.126 0.065
Note: 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

 

 

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.

 

Table 2: Biodiversity analysis of Seed mycoflora in maize by Blotter plate method
Zone II
Ct Species Dn F

(In %)

Ab RD

(In %)

RF

(In %)

RA

(In %)

IVI

(In %)

D=Pi*Pi H=-{(pi) × ln(pi)} E={H/ln(S)}
OS Aspergillus flavus 4.700 100.000 0.350 21.211 24.691 35.009 80.912 0.0727 0.353 0.170
Aspergillus niger 5.850 50.000 0.218 26.401 12.346 21.788 60.534 0.0407 0.323 0.155
Rhizopus stolonifer 2.833 90.000 0.190 12.787 22.222 18.994 54.003 0.0324 0.309 0.148
Fusarium verticilioides 1.800 75.000 0.101 8.123 18.519 10.056 36.698 0.0150 0.257 0.124
Alternaria alternata 2.600 25.000 0.048 11.734 6.173 4.842 22.748 0.0057 0.196 0.094
Penicillium  notatum 2.250 40.000 0.067 10.154 9.877 6.704 26.735 0.0079 0.215 0.104
Macrophomina phaseolina 1.625 20.000 0.024 7.334 4.938 2.421 14.693 0.0024 0.148 0.071
Rhizoctonia soloni 0.500 5.000 0.002 2.256 1.235 0.186 3.677 0.0002 0.054 0.026
PDS Aspergillus flavus 4.500 100.000 0.342 19.268 23.256 34.221 76.744 0.0654 0.349 0.159
Aspergillus niger 4.100 50.000 0.156 17.555 11.628 15.589 44.772 0.0223 0.284 0.129
Rhizopus stolonifer 2.605 95.000 0.188 11.155 22.093 18.821 52.069 0.0301 0.304 0.138
Fusarium verticilioides 3.267 75.000 0.186 13.987 17.442 18.631 50.060 0.0278 0.299 0.136
Alternaria alternata 1.500 10.000 0.011 6.423 2.326 1.141 9.889 0.0011 0.112 0.051
Penicillium  notatum 1.083 60.000 0.049 4.638 13.953 4.943 23.535 0.0062 0.200 0.091
Macrophomina phaseolina 2.300 25.000 0.044 9.848 5.814 4.373 20.034 0.0045 0.181 0.082
Curvularia lunata 2.000 10.000 0.015 8.563 2.326 1.521 12.410 0.0017 0.132 0.060
Bipolaris maydis 2.000 5.000 0.008 8.563 1.163 0.760 10.487 0.0012 0.117 0.053
DS Aspergillus flavus 5.225 100.000 0.407 26.381 23.810 40.661 90.852 0.0917 0.362 0.174
Aspergillus niger 3.700 50.000 0.144 18.681 11.905 14.397 44.983 0.0225 0.285 0.137
Rhizopus stolonifer 2.850 100.000 0.222 14.390 23.810 22.179 60.378 0.0405 0.323 0.155
Fusarium verticilioides 1.714 70.000 0.093 8.655 16.667 9.339 34.661 0.0133 0.249 0.120
Alternaria alternata 1.250 20.000 0.019 6.311 4.762 1.946 13.019 0.0019 0.136 0.065
Penicillium  notatum 2.167 45.000 0.076 10.939 10.714 7.588 29.241 0.0095 0.227 0.109
Macrophomina phaseolina 1.400 25.000 0.027 7.069 5.952 2.724 15.745 0.0028 0.155 0.074
Curvularia lunata 1.500 10.000 0.012 7.573 2.381 1.167 11.122 0.0014 0.122 0.059
Note: 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

 

Elham et al., [28] recorded percentage frequency and relative density the members of genus Fusarium spp. were predominantly isolated from maize grains as internal  mycoflora at all locations ( Fr. range 8.0 – 10% and R.D. 2.5 -3.5  as external mycoflora and internal mycoflora Fr .22.1 – 45% and  R.D. 10.8 – 25% ) . The second most prevalent genus as internal mycoflora was Alternaria spp. (Fr.20 -27.5% and RD. 10.25 -17.5%) as external mycoflora for internal mycoflora (Fr. 35-45% and R.D. 20%). The most predominant external mycoflora of the mold was Aspergillus spp. (Fr.27.5- 37.5 and R.D.15.13 – 23.8%) and for internal mycoflora relative density and frequency were slightly low (Fr. 16 – 18.4% and R.D. 12 – 15.3%). Penicillium sp. recorded the lowest value of external and internal mycoflora.

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 21.7-33.2×103 CFU/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] 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.

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.

Ghiasian et al. [32] showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus.

 

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.

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