Research Article

Characterization of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus by Cultural and Immunological Methods

Iftikhar Jan* and Sahib Alam

Department of Agricultural Chemistry and Biochemistry, the University of Agriculture, Peshawar, 25130, Pakistan.

Received | March 21, 2021; Accepted | June 17, 2021; Published | August 03, 2021

*Correspondence | Iftikhar Jan, Department of Agricultural Chemistry and Biochemistry, the University of Agriculture, Peshawar, 25130, Pakistan; Email: ijan719@gmail.com

Citation | Jan, I. and S. Alam. 2021. Characterization of aflatoxigenic and nonaflatoxigenic isolates of Aspergillus flavus by cultural and immunological methods. Sarhad Journal of Agriculture, 37(3): 1062-1072.

DOI | https://dx.doi.org/10.17582/journal.sja/2021/37.3.1062.1072

Keywords | Aflatoxins, Maize, Soil, Aflatoxigenic, Aspergillus flavus, ELISA

Introduction

Aflatoxins represent a major class of mycotoxin that are highly toxic and carcinogenic in nature. They are mostly produced by closely related species of Aspergillus in many agricultural products worldwide (Pickova et al., 2021). The B-group aflatoxins i.e., AFB1 and AFB2 are typically produced by Aspergillus flavus, while most toxigenic strains of Aspergillus parasiticus have great potential in producing all the four kinds i.e., AFB1, AFB2, AFG1 and AFG2 of aflatoxins (Dorner, 2004). Aflatoxins are chemical derivatives of difuranocoumarin formed by fusion of two furans and one coumarin ring synthesized by polyketide pathway. Aflatoxins are classified into more than eighteen different types among which AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2 are the most important and naturally occurring members found in different food commodities (Benkerroum, 2020). Many species in section Flavi also produce a wide range of other mycotoxins besides aflatoxin like paspaline, aspergillic acid, cyclopiazonic acid (CPA) and kojic acid (Frisvad and Samson, 2000).

Aflatoxin causes aflatoxicosis was first reported in England during 1960s after the outbreak of ‘Turkey X’ disease which killed several thousand young Turkeys upon eating aflatoxin contaminated peanuts (Blount, 1961). Later on, same sort of incidence was investigated in ducklings, chickens, swine and calves (Asplin and Carnaghan, 1961; Loosmore and Harding, 1961; Harding et al., 1963). In 1961, mycotoxin producing mold was recognized as A. flavus, while the toxin was given the name “aflatoxin” by virtue of its origin (Ellis et al., 1991). A later study found that CPA was also present in peanut samples from the feed implicated in the outbreak. This latter observation proposed that CPA might have played an important role in the epidemic of Turkey X disease (Cole, 1986).

Aflatoxins are carcinogenic, mutagenic, immunosuppressive and growth inhibitory, affecting humans, livestock, poultry and wildlife (Tola and Kebede, 2016). The lethal effects of aflatoxins fluctuate and depend on ingested dose, toxin type, exposure duration, mode of action and defense mechanism of an organism. The disease known as aflatoxicosis caused by aflatoxins ingestion are predominantly linked with both acute and chronic toxicity in human and in animal populations (Bennett and Klich, 2003). The outbreak of acute aflatoxicosis in human was characterized by nausea, vomiting, abdominal pain, pulmonary edema, unconsciousness, convulsions and even death (Mwanda et al., 2005). However, chronic exposure to low doses may lead to liver cancer development (Claeys et al., 2020). Intake of aflatoxin can cause serious illness in animal particularly gastrointestinal disorders, reduced reproduction, alleviate milk and egg production, anemia and cancer of liver, colon and kidney (Peles et al., 2019). The International Agency of Research on Cancer (IARC) has categorized AFB1 and AFM1 as Group-1 and Group-2B human carcinogen because of high menace for hepatocellular carcinoma in individuals exposed to aflatoxins (Ostry et al., 2017).

Aflatoxin contamination is primarily observed in different agricultural commodities including corn, cereals, soybeans, sorghum, peanuts and other food and feed crops (Alshannaq and Yu, 2017). In many less developed countries aflatoxin is a common food contaminant particularly in their staple diets. It has been reported that the consumption of aflatoxin contaminated food in India and Kenya caused aflatoxicosis which accounted for the death of 100 and 125 people, respectively (Moss, 2002; Lewis et al., 2005). Therefore, the United States Food and Drug Administration (USFDA) has settled an allowable limit of total aflatoxin in corn destined for use as human food and animal feed at 20 ppb, while the guidelines in the European Union (EU) tolerate no more than 4 ppb of total aflatoxins (Van Egmond and Jonker, 2003). Any products found to have aflatoxins above the recommended limits are usually rejected for subsequent marketing. The US market losses from aflatoxin contamination alone are estimated to be in the hundreds of billions (Wu and Guclu, 2012). Due to molds and mycotoxins contamination, there is a huge pressure on Pakistan economy by rejection of shipment of agricultural produces in the international market. In this regard it appears very critical that research on detection and control of aflatoxins in food should be undertaken on priority basis in order to enable Pakistan to produce products comparable to World Trade Organization (WTO) standards as well as to safeguard its citizens from these toxic contaminants.

It is pertinent to mention that not all strains of A. flavus produce aflatoxin and this prompts the adoption of multiple screening techniques to find out their real aflatoxigenic potential. The nonaflatoxigenic strains could be used to compete the aflatoxigenic strains in the field or stored grains for ecological niches. Thus, it is hypothesized that the A. flavus flora of different agroecological zones of Khyber Pakhtunkhwa province of Pakistan may have potential nonaflatoxigenic strains that could be used for the control of aflatoxigenic ones. Therefore, this study was carried out to (1) determine the occurrence frequency of A. flavus population in soil and stored maize grain samples collected from various parts of Khyber Pakhtunkhwa and to (2) characterize the isolated A. flavus strains into aflatoxigenic and nonaflatoxigenic by employing cultural and immunological techniques.

Materials and Methods

Samples collection

Samples of stored maize grain from farmer premises and soil of agricultural fields (0.5 kg each) were collected from four agroecological zones (AEZs) i.e., AEZ-A (Swat (SWT), Dir (DIR) and Buner (BUN)), AEZ-B (Abbottabad (ABT), Mansehra (MAN) and Haripur (HAR)), AEZ-C (Charsadda (CHD), Mardan (MAR) and Peshawar (PES)) and AEZ-D (Tank (TAN), Dera Ismail Khan (DIK) and Lakki Marwat (LMT) districts) of Khyber Pakhtunkhwa (KP) province of Pakistan (Figure 1). The samples were collected from six locations in each district. A total of 144 samples (36 samples per zone) were collected in clean polyethylene Ziplock bags, shipped to laboratory and screened for Aspergillus Section Flavi strains.

 

Isolation and identification of Aspergillus section Flavi isolates

The soil and maize samples were screened on potato dextrose agar (PDA) medium to isolate Aspergillus section Flavi isolates using dilution plate technique (Rosa et al., 2006; Shah et al., 2010). In this method 1.0 g of each grounded sample was dissolved in 10 mL sterilized distilled water in glass Universal bottles containing 0.01% Tween 80. The content was mixed thoroughly and serial dilution (up to 4 times) were prepared by successively transferring 1 mL suspension to 9 mL sterilized distilled water. A 100 μL aliquot of suspension from 10-4 dilution was transferred to PDA media in Petri plates and seeded on the whole surface by using sterilized wire loop. All the Petri plates were incubated at 25 ºC for 7-10 days. The pure cultures of Aspergillus section Flavi isolates were obtained by single spore culture technique. The isolates were morphologically identified on the basis of their macroscopic (mycelium color, colony reverse color and formation of sclerotia) and microscopic characteristics (conidiophores, vesicles, matulae, phialides and conidia) according to recommended synoptic key descriptions (Clayton, 1977; Klich, 2002; McClenny, 2005). The isolates were further cultured on Aspergillus Flavus Parasiticus Agar (AFPA) medium to confirm their identity as A. flavus and A. parasiticus strains (Pitt et al., 1983).

Selection of nonaflatoxigenic strains

All the Aspergillus section Flavi isolates were screened for aflatoxin production through cultural and immunological methods.

Cultural methods

The following cultural methods were employed for the identification of nonaflatoxigenic isolates.

Production of blue fluorescence on coconut cream agar (CCA)

In this technique a small, pointed inoculum of Aspergillus section Flavi was placed on 50% coconut cream agar medium (Dyer and McCammon, 1994). The Petri plates were incubated at 25 ºC for 7 days after inoculation. All aflatoxigenic isolates emitted characteristic blue fluorescence on the reverse side of CCA medium plates under long-wave ultraviolet (UV) light (365nm) whereas the nonaflatoxigenic isolates did not produce any fluorescence under the same wavelength.

Room temperature phosphorescence

The aflatoxins production ability was further confirmed by growing each isolate on sterilized malt extract agar (MEA) medium modified with β-cyclodextrin (0.3%) and sodium deoxycholate (0.6%). A small portion of the actively growing part of colony of each isolate was inoculated in the center of modified MEA plate and incubated upright at 25 ºC. The Petri plates were examined for room temperature phosphorescence on day 5th of incubation period. The plates were examined in such a way that they were placed upright over UV lamp. The appearance of bright green phosphorescence for a while represented positive indication of aflatoxigenicity after turning off the excitation source (Rojas-Duran et al., 2007).

Ammonium hydroxide vapor-induced color change

A single colony of Aspergillus isolates was inoculated in the center of PDA medium plates and incubated in the dark at 28 °C for seven days or until the fungal mats covered the Petri dish. The dishes were inverted, and concentrated ammonium hydroxide solution was sprayed on inside of the lids so that fungal mats were fully exposed to the vapors. The appearance of plum-red color on underside of cultural plate indicated aflatoxin production ability of isolate (Abbas et al., 2004).

Sclerotia formation

The isolates were further assessed for their aflatoxigenicity on the basis of sclerotia formation on NaNO3 added Czapek yeast agar (CYA) medium. The isolates were cultured on the media for 21 days at 28 °C. The isolates which did not produce sclerotia were characterized as nonaflatoxigenic and those that produced sclerotia were marked as aflatoxigenic (Abbas et al., 2005).

Immunological method

The nonaflatoxigenicity of selected isolates (n= 30) were further confirmed through enzyme linked immunosorbent assay (ELISA) using commercially available test kit RIDASCREEN® Aflatoxin Total (R-Biopharm AG, Darmstadt, Germany) according to the manufacturer instruction. The selected isolates were cultured on corn meal agar (CMA) medium for 10 days at 30oC. An agar plug (2 g) from the cultural medium was extracted with 10 mL of 70% methanol for 10 minutes at room temperature on high-speed reciprocal shaker. The extracts were centrifuged (10 min 3500g-1), diluted with water and then analyzed by ELISA. The absorbance of spiked and test samples were measured by spectrophotometer at wavelength of 450 nm. Concentration of aflatoxin was calculated from calibration curve which was obtained using aflatoxin standards with the following concentrations: 0, 5, 15, 45, 135 and 405 ng L-1. In this assay limit of detection for total aflatoxin was approximately 1.75 ng L-1.

Statistical analysis

The data regarding cultural techniques and determination of aflatoxin were subjected to analysis of variance (ANOVA) and least significant difference (LSD) test at 0.05 level of probability by using the statistical software package Statistix 8.1.

Results and Discussion

Isolation, identification and geographical distribution of Aspergillus section Flavi

A total of 434 isolates of Aspergillus section Flavi were isolated from soil and maize samples collected from four agroecological zones of KP province of Pakistan. The isolates were putatively identified as A. flavus on the basis of morphological characteristics and their orange red colored formation on reverse side of AFPA medium plates (Figure 2). Among all the isolates, 236 were obtained from soil samples and 198 from maize grains (Figure 3). In case of sample locations, the highest total member of section Flavi were isolated from samples collected from districts CHD (n = 44) and DIK (n = 42) while the lowest number were found in district BUN (n = 32). Similarly, maximum (n = 25) number of section Flavi isolates among all soil samples was recorded in DIK while their minimum (n = 16) population occurred in district HAR, ABT and TAN. In case of maize samples, BUN showed the highest (n = 22) while HAR possessed the lowest (n = 12) number of section Flavi isolates. Across all the four AEZ, the highest population of Aspergillus section Flavi was observed in AEZ-C (n = 115) followed by AEZ-D (n = 113), AEZ-A (n = 107) and AEZ-B (n = 99). Apart from Aspergillus section Flavi, colonies of other molds were also observed in soil and maize samples (Figure 4). In soil samples, Pencillium spp. (31.28%) and Rhizopus (17.25%) ranked the highest while Alternaria spp (4.63%) was the lowest among all the isolated fungi. Likewise, maize samples had high relative occurrence of Fusarium spp (26.0%) and Paecilomyces spp (22.67%) but less incidence of Cladosporium spp (2.33%).

 

.

 

Detection of nonaflatoxigenic aspergillus isolates by cultural techniques

The blue fluorescence technique revealed that 71.76% of all the A. flavus isolates isolated from maize grain samples and 66.04% of the soil dwelling isolates were aflatoxigenic in nature (Figure 5). Among all soil samples, PES district of AEZ-C had abundant (87.07%) while DIR district of AEZ-A had the least (53.33%) BF emitting isolates. In case of maize samples, most BF producers population occurred in district MAR (85.42%) of AEZ-C, however their lowest percentage was examined in district BUN (54.55%) of AEZ-A. Across all the zones, AEZ-C (78.92%) showed more BF producer’s isolates than AEZ-B (70.73%), AEZ-D (64.53.09%) and AEZ-A (61.41%).

 

Likewise, significantly higher percentage of RTP emitting isolates were examined in maize (64.71%) as compared to soil (66.04%) samples (Figure 6). Among all soil samples, MAN and MAR districts of AEZ-B and AEZ-C had abundant (80.0% and 77.78%, respectively) while BUN district of AEZ-A had the lowest (42.59%) RTP producing isolates. Regarding maize samples, abundant RTP emitting isolates were noted in ABT (70.79%) and CHD (70.0%) districts of AEZ-B and AEZ-C, whereas SWT region of AEZ-A had less RTP producing isolates (43.59%). Among all zones, the highest population of phosphorescence producing isolates were found in AEZ-B (65.80%) followed AEZ-C (63.36%) AEZ-D (57.09%) and AEZ-A (55.10%).

 

The results of ammonium hydroxide vapor-induced (AV) induced color change (Figure 7) showed that AEZ-C had the highest aflatoxigenic isolates (87.71%) followed by AEZ-B (81.42%) while the lowest population was recorded for AEZ-D (75.80%). Regarding sample sources, promising toxigenic Aspergillus isolates were found in maize (82.40%) as compared to soil (78.18%) samples. Among all the soil samples, the highest aflatoxigenic isolates were found in the samples from PES (87.04%) district of AEZ-C followed by SWT (85.96%) of AEZ-A, whereas fewer isolates were noted from TAN (64.58%) district of AEZ-D. In case of maize samples, abundant toxigenic isolates were observed in PES (93.75%) and CHD (90.0%) districts of AEZ-C, while the lowest number were examined in DIK (64.71%) district of AEZ-D.

 

The aflatoxigenicity of Aspergillus isolates were also confirmed on the basis of their sclerotia formation on CYA medium (Figure 8). Among all the zones, AEZ-D contained the highest sclerotia forming isolates (35.52%) as compared to AEZ-B (31.31%), AEZ-C (28.84%) and AEZ-A (26.33%). It was observed that more sclerotia forming isolates were present in soil (40.15%) than in maize (20.85%) samples. Among all the soil samples, TAN district of AEZ-D possessed comparatively higher (58.33%) population of sclerotia forming isolates followed by LMT (52.17%) of AEZ-D, whereas BUN district of AEZ-A had the least (18.52%) isolates. In all maize samples, the highest population of Aspergillus that developed sclerotia were recorded for DIK (43.14%) of AEZ-D and BUN (33.33%) of AEZ-A, while the lowest percentage was observed for LMT (10.26%) district of AEZ-D.

 

The comparative analysis of all four cultural techniques divulged that soil had overall higher mean toxigenic isolates (62.27%) than maize grain samples (57.74%). Further observation showed that among all soil samples, MAN district of AEZ-B had the highest (71.67%) and BUN district of AEZ-A had the lowest (49.07%) average number of aflatoxigenic isolates. In case of maize samples, ABT district of AEZ-B was found to possess a mean higher number (67.16%) of toxigenic Aspergillus section Flavi isolates, while SWT district of AEZ-A had relatively less number of toxigenic isolates (50.64%). Regarding sample locations, maximum average aflatoxigenic Aspergillus section Flavi isolates were found in MAR (65.71%) and CHD (64.41%) districts followed by MAN (64.31%), while less aflatoxigenic isolates (52.57%) were examined in maize grain samples from BUN district (Table 1). The findings (Table 2) further revealed that AEZ-C had higher (64.84%) mean aflatoxigenic isolates than AEZ-B (62.32%), AEZ-D (58.37%) and AEZ-A (54.77%). With respect to cultural techniques, all the four methods successfully differentiated aflatoxigenic and nonaflatoxigenic isolates, however NH4OH vapor induced color change of the reverse side of colonies was found more efficient (80.29%) than RTP (68.90%), blue fluorescence (60.40%) and sclerotia formation (30.50%).

 

Table 1: Occurrence (%) of aflatoxigenic Aspergillus section Flavi isolates in stored maize grain and soil samples collected from different Agroecological zones (AEZs) of Khyber Pakhtunkhwa.

Agroecological zone	Location/District	Soil	Maize	Mean
AEZ-A	SWT	62.28	50.64	56.46def
	BUN	49.07	56.06	52.57f
	DIR	56.67	53.89	55.28ef
AEZ-B	HAR	68.23	56.25	62.24abc
	MAN	71.67	56.94	64.31ab
	ABT	53.65	67.16	60.40bcd
AEZ-C	CHD	64.24	64.58	64.41ab
	MAR	69.44	61.98	65.71a
	PES	67.56	60.42	64.0ab
AEZ-D	DIK	65.33	51.96	58.65cde
	TAN	56.77	57.89	57.33de
	LMT	62.32	55.13	58.72cde
Mean	-	62.27a	57.74b	-

Mean values in row and column followed by different alphabets are statistically significant at p<0.05.

 

Table 2: Detection of aflatoxigenic isolates (%) of Aspergillus section Flavi using cultural techniques of blue fluorescence (BF), room temperature phosphorescence (RTP), ammonium hydroxide vapor induced color change (AV) and sclerotia formation (SF) on CCA, modified MEA, PDA and CYA medium.

Origin of A. flavus isolate	Cultural technique				Mean
	BF	RTP	AV	SF
AEZ-A	61.41	55.10	76.24	26.33	54.77c
AEZ-B	70.73	65.80	81.42	31.31	62.32a
AEZ-C	78.92	63.36	87.71	28.84	64.71a
AEZ-D	64.53	57.09	75.80	35.52	58.37b
Mean	68.90b	60.34c	80.29a	30.50d

Mean values in row and column assigned with identical alphabets are statistically non-significant at p<0.05.

 

Detection of nonaflatoxigenic Aspergillus isolates by immunological technique

The ELISA results showed that the highest total aflatoxins content (230.78 μg Kg-1) was recorded for isolate labeled as AFS32 whereas no aflatoxins were produced by isolates AFS5, AFS17, AFS25 and AFS33. In this technique, the recovery rate of total aflatoxins was between 95.50-105.42% with relative standard deviation (RSD) value ranged from 3.65-4.68% (Table 3).

 

Table 3: Aflatoxin level (µg Kg-1) of selected A. flavus isolates determined by ELISA.

S. No.	A. flavus isolate	Total aflatoxin (µg Kg-1)	S. No.	A. flavus isolate	Total aflatoxin (µg Kg-1)
1.	AFS1	146.37±1.76	16.	AFS25	0.00
2.	AFS2	170.58±2.08	17.	AFS26	192.48±2.65
3.	AFS3	55.55±0.90	18.	AFS27	142.18±2.34
4.	AFS5	0.00	19.	AFS29	160.86±1.22
5.	AFS6	158.38±1.49	20.	AFS31	95.65±1.25
6.	AFS7	184.51±2.10	21.	AFS32	230.78±1.54
7.	AFS9	130.22±2.48	22.	AFS33	0.00
8.	AFS10	84.55±1.16	23.	AFS35	130.44±2.91
9.	AFS12	65.34±2.24	24.	AFS36	198.54±2.14
10.	AFS13	210.62±2.25	25.	AFS39	110.46±1.49
11.	AFS17	0.00	26.	AFS43	6.43±0.57
12.	AFS18	50.86±1.10	27.	AFS44	226.22±2.58
13.	AFS19	31.37±0.72	28.	AFS45	175.77±1.18
14.	AFS21	165.72±2.33	29.	AFS47	220.42±2.24
15.	AFS23	112.37±1.62	30.	AFS49	168.36±1.89

Each value is mean of three replications ± standard deviation.

 

In this study, the population diversity and geographic distribution pattern of Aspergillus section Flavi were determined to identify indigenous nonaflatoxigenic strains as potent biocontrol candidate for prevention of aflatoxin contamination in agricultural crops. A total of 434 isolates of Aspergillus section Flavi were isolated from field soil and maize grains collected from farmers’ storage structures across four agroecological zones in Khyber Pakhtunkhwa province of Pakistan. All soil and maize samples contained Aspergillus section Flavi isolates, though abundant population was observed in soil samples. Additionally, the incidence of section Flavi varied widely among districts and AEZ, however, the prevalence of Aspergillus was more consistent in the southern region i.e., AEZ-C and AEZ-D than in the northern region i.e., AEZ-A and AEZ-B. The climatic conditions of the southern part of Khyber Pakhtunkhwa ranges from warm and sub-humid in Zone C to hot and arid in Zone D. Similarly, drought stress, less rainfall and high temperature was more common in the southern region as compared to the northern part (EPA, 2016). All these factors and many other micro-climatic factors usually contributed in higher population of Aspergillus section Flavi in both AEZ-C and AEZ-D. A comparative study conducted in major almond producing areas of California, USA, showed higher frequencies of A. flavus L strains in the southern region as compared with the northern region (Donner et al., 2015). Similar results associated with high predominance of A. flavus in the soil ecosystem were reported from West Africa, Nigeria and Thailand (Cardwell and Cotty, 2002; Ehrlich et al., 2007; Donner et al., 2009). Likewise, fluctuation in prevailing environmental conditions of temperature, rainfall and relative humidity had significant influence on the distribution, density and structure of aflatoxin producing fungi as well as vulnerability of crop plants to such communities (Van der Fels-Klerx et al., 2016; Mmongoyo et al., 2017).

In Pakistan, huge amount of maize grains are generally stored for extended time period under improper and insufficient storage conditions which consequently result in degradation of the grains quality because of pest attack and mold infestation. Therefore, in this research work we underlined the incidence and prevalence of aflatoxigenic fungal isolates in stored maize grains samples collected from traditional farmer’s storage structures across the four AEZ of KP. The data obtained in this study divulged that all stored maize grain samples were at risk of fungal infestation and mycotoxins contamination. The data obtained showed that all grains were primarily contaminated with Aspergillus section Flavi isolates, though co-contamination with many field and storage fungi occurred in most of the samples. The analysis further indicated high predominance of some toxigenic fungal genera including Aspergillus, Fusarium, Paecilomyces and Penicillium as compared with other fungal isolates. This widespread distribution and prevalence of Aspergillus section Flavi in stored maize were observed previously in China (Xing et al., 2017), Nigeria (Muhammad et al., 2019) and Spain (Alborch et al., 2012). The development of these fungi could be affected by moisture content of the product (Gtorni et al., 2009). However, some other factors like temperature, water activity (Alam et al., 2009), physical condition of the grains (Hocking, 1991) and post-harvest management practices (Hell and Mutegi, 2011; Akowuah et al., 2015) also support fungal growth in maize grain during storage.

The cultural methods used for the detection of aflatoxins were applied to 434 members of section Flavi included blue fluorescence, RTP phosphorescence, NH4OH vapor induced color change and sclerotia formation. The data obtained from combined cultural techniques showed high (60.0%) frequency of aflatoxigenic A. flavus than nonaflatoxigenic (40.0%) isolates with reference to substrates types and geographical origin. A significant (P<0.05) higher aflatoxin producers isolates of A. flavus were observed in soil than in maize samples. The relative proportion of toxigenic and atoxigenic strains of A. flavus varied significantly (p<0.05) among the districts sampled during the survey. Across districts, the highest prevalence of aflatoxin producers was observed in Mardan, Charsadda and Mansehra, while the lowest occurrence of aflatoxigenic strains was noted in district Buner. Similarly, the frequencies of aflatoxigenic isolates were significantly (P<0.05) higher than that of nonaflatoxigenic isolates in all the four AEZs. The incidence of aflatoxigenic isolates in AEZ-C and AEZ-B were comparatively greater than that of aflatoxigenic strains in AEZ-A and AEZ-D. The present data finds supportive evidence from previous investigation conducted in USA, and Italy which showed high proportion of aflatoxigenic isolates in soil and maize samples than nonaflatoxigenic isolates (Giorni et al., 2007; Horn and Dorner, 1999). The relative proportion of aflatoxigenic and nonaflatoxigenic isolates of A. flavus observed in this study was influenced by many factors such as geographical regions, soil condition, soil type, crop sequence history, crop management practices, day length, insect presence, fungicide application history and irrigation source and frequency (Orum et al., 1997).

In the course of this study we also compared the efficacy of four cultural techniques used for differentiation of aflatoxigenic and nonaflatoxigenic isolates of A. flavus. The current findings confirmed that NH4OH vapor induced color change had greater (80.29%) potential in the distinction of aflatoxigenic isolates followed by blue fluorescence (68.90%) and RTP (68.90%). Moreover, 30.50% of the total isolates produced sclerotia was considered as positive isolates with respect to their aflatoxin production. It is evident from the present results that the occurrence of blue fluorescence, emission of RTP, orange-red pigment formation of isolates after exposure to NH4OH vapor as well as formation of sclerotia were strongly an indication of aflatoxin producing ability of the isolates. These findings were in concordance to the previously published reports (Abbas et al., 2004, 2005; Rojas-Duran et al., 2007).

This study also presents a distinctive method for direct extraction of aflatoxin from solid nutrient medium and their simultaneous quantification by ELISA. The highly specialized and sensitive method of ELISA was applied on selected 30 out of 434 isolates for their aflatoxins production in CMA medium. The ability of A. flavus to produce total aflatoxins varied significantly (p<0.05) among different strains. The results showed that AFS32, AFS44, AFS47 and AFS13 produced greater amount of total aflatoxin (>200µg Kg-1) as compared with other isolates. The isolates AFS5, AFS17, AFS25 and AFS33 produced no aflatoxins when analyzed with ELISA. The variation among aflatoxigenic Aspergillus isolates in production of aflatoxins determined in the present study had also been reported previously (Fraga et al., 2007).

Conclusions and Recommendations

The isolates AFS5, AFS17, AFS25 and AFS33 confirmed for their nonaflatoxigenicity by using cultural and ELISA methods were considered as isolates of interest and could be used as candidate biocontrol agents for prevention of pre-harvest aflatoxin production in different crops. It was also suggested that cultural and immunological techniques should be used in combination with analytical and molecular techniques for authentic segregation of nonaflatoxigenic isolates of A. flavus from aflatoxigenic isolates.

Acknowledgements

We are thankful to the Higher Education Commission (HEC) of Pakistan for providing financial grant under the Project No. 8688/KPK/NRPU/R&D/HEC/2017. Thanks, are also extended to the University of Liverpool, UK for research facilitation to successfully complete this study.

Novelty Statement

This is the first study of its type in Khyber Pakhtunkhwa to explore indigenous Aspergillus flavus flora for nonaflatoxigenic strains to be used as biocontrol agents for prevention of aflatoxin contamination in different crops.

Author’s Contribution

Iftikhar Jan: Conducted the experimental work and wrote the manuscript.

Sahib Alam: Designed and supervised the study.

Conflict of interest

The authors declare no conflict of interest.

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