Exploration of aflatoxigenic potential and seed colonization of Aspergillus flavus in sunflower

 

Vikas Verma Patel¹*, Saurabh Kumar², Nagendra Prasad³

¹Assistant Professor, Department of Botany, V.R.A.L. Government Girls Degree College, Bareilly- 243001

M.J.P. Rohilkhand University, Bareilly-243001, U.P., India.

²Assistant Professor, Department of Botany, D.A.V. (PG) College, Muzaffarnagar, U.P., India- 251001.

³Birbal Sahni Institute of Palaeosciences, 53-University Road, Lucknow-226007, U.P., India.

 

ABSTRACT:

Aflatoxin, a mycotoxin found commonly in maize, peanuts, and sunflower worldwide, is associated with liver cancer, acute toxicosis, and growth impairment in humans and animals. In India, sunflower seeds are a source of snacks, cooking oil, and animal feed. These seeds are a potential source of aflatoxin contamination. However, reports on aflatoxin contamination in sunflower seeds and cakes are scarce. The objective of the current study was to determine the potential of Aspergillus flavus and total aflatoxin concentrations in sunflower seeds and cakes from small-scale oil processors in Rohilkhand region of Uttar Pradesh. 126 cultures of Aspergillus flavus were obtained from sunflower kernels, which showed wide variation in their cultural characters. Colour of conidial heads being a stable character was used to divide them into four groups out of which two representative isolates each, based on minimum and maximum number of sclerotia cm^-2 of the culture medium were selected. Aflatoxin production by an isolate had positive correlation with the sclerotia production. These results can help in identifying the potent of A. flavus isolates for aflatoxin production and developing proper management strategies. In summary, humans and animals are potentially at high risk of exposure to aflatoxins through sunflower seeds and cakes from micro-scale millers in India and location influences risk.

 

 

 

INTRODUCTION: 

Infection of sunflower (Helianthus annuus L.) kernels with Aspergillus flavus is significantly important because, apart from seed borne diseases, the fungus has potential to produce toxins. Species of Aspergillus are common mycotoxigenic fungi predominantly associated with heavy loss of food and feedstuffs during storage worldwide. First time these toxins were identified as aflatoxins by Asao et al.¹, which were later reported as potent carcinogens². Sunflower seeds were shown to support aflatoxin production³, this was first reported by the study of toxin production in five varieties of sunflower seeds.

 

Adaptability of A. flavus to varied agroclimatic conditions and extremely wide host range has possibly resulted in the existence of its different isolates. Different isolates were categorized based on their colony characters⁴. Screening of aflatoxigenic property of some Aspergillus flavus isolated from sunflower seeds and its products⁵, reported as it is not necessary that all the isolates of A. flavus produce aflatoxins. Several studies carried out on variability in aflatoxin production potential of A. flavus isolates from different cereal crops^6,7. However, no relation between cultural characters and aflatoxin production has been established so far. Aspergilli are well known to invade different agricultural commodities in the pre and post-harvest stages^8,9. They apparently damage grain quality, decrease nutritional value, weight, and palatability. Potent danger of this fungus is based mainly on two factors. First, the penetration of kernels by fungus and second, the aflatoxin production in the kernels of sunflower seeds. It can be useful if potential of the fungus related to these factors can be determined before the fungus infects sunflower kernels. This can help in developing a proper management strategy, indicate the possible use of botanical extracts of different natural wild plants as alternative agents for developing plant-based preservatives against post-harvest fungal infestation of food commodities and aflatoxin contaminations¹⁰. Recently, the molecular mechanism of aflatoxins biosynthesis revealed that the genes involved in the production of aflatoxins are located in a co-regulated gene cluster that encodes two regulatory proteins i.e., aflR and aflJ¹¹. A comparative study of the quality assessment of Vitex negundo leaves from different regions explained and reported that the estimation of physio-chemical parameters, heavy metals and aflatoxin is highly essential for raw drugs or plant parts used for the production of chemical compounds¹². Aspergillus flavus present in sunflower seeds were isolated and tested for their aflatoxin content¹³. The presence of mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds used for human consumption were also identified¹⁴. In the present investigation, an attempt has been made to study aflatoxin production potential of A. flavus isolates based on their cultural characters. A relation between aflatoxin potential and seed colonization in post-harvest sunflower seeds has also been studied.

 

MATERIALS AND METHODS:

Cultural studies: A total of 126 cultures of A. flavus were obtained from kernels of sunflower seeds obtained from 8 different locations from the districts of Bareilly, Pilibhit, Moradabad and Shahjahanpur in Uttar Pradesh state after the harvest. Isolation was done by plating 100 surface sterilized kernels from each of the 8 seed lots, on to PDA and incubated at 25± 1⁰C for 7days. All the A. flavus cultures were purified by single spore isolation and grown on 0.7% YES+ salt medium¹⁵ for 10 days to record texture of mycelial mat and sporulation, and on PDA to record sclerotia production after 30 days inoculation. These characters were categorized as colour of conidial heads: olive green (hue 5 Y 5/4), green (hue 2.5 Y 5/6), olive yellow (hue 5 Y 7/8) and yellow (hue 5Y 7/8) and sclerotia production cm^-2 of culture: nil, scanty (1-5) moderate (6-10) and abundant (>10).

 

All the cultures were divided into 4 groups (Af- 1 to Af- 4) based on colour of the conidial heads. From each group, two representative isolates were selected for future studies, based on maximum and minimum number of sclerotia produced by the cultures within that group. For different experiments, each isolate was replicated thrice.

 

Seed colonization:

Twenty gm apparently healthy seeds were washed with water, surface sterilized with 0.1% HgCl₂ for 2-3 min and rinsed twice with sterilized distilled water. One ml of spore suspension (4×10⁶ ml^-1) of 8 days old A. flavus cultures on PDA was added to each 20-gm seed lot in sterile Petri plates. They were swirled gently to spread spore suspension uniformly and incubated at 25± 1^º C under 100% RH for 8-days. Observations on percent seed colonization were recorded.

 

Aflatoxin estimation:

Aflatoxin was extracted from the filtrates of 10-days old cultures grown on 0.7% YES + salt medium according to the method of Pons et al. ¹⁶. Estimation was done by TLC using silica gel-C coated plates run in CHCl₃: C₂H₅OH (95:5 v/v) solvent system. These plates were observed under UV light at 363 nm. All the culture filtrates produced blue, fluorescent spots corresponding to standard aflatoxin B1. These spots were eluted in cold methanol, diluted to 5 ml and OD was determined by using Beckman, DU-7 spectrophotometer at 363 nm. Aflatoxin concentration was calculated from the standard curve.

 

RESULTS AND DISCUSSION:

Categorization of A. flavus and aflatoxin production:

Among the groups of A. flavus there was variation in colony texture, sclerotia production and colour of conidial heads (Table 1). However, within a group, colour of conidial heads and sclerotial production by an isolate did not very significantly after successive sub-culturing during the course of whole investigation. Dry weight of the mycelium and defined quantity of the medium was variable. Rate of radial growth was variable i.e., 10.4 to 19.0 mm/24 hrs. in all the isolates (Table 2). Isolates Af-1-(1 and 2) produced aflatoxin 394 and 666 ppb. Sclerotial production was absent in Af-1-1 or scanty in Af-1-2 and mycelial mat was plane or slight wrinkled. Isolates A-2-(1 and 2) had plane mycelial mat and sclerotial production was moderate and scanty i.e., it varied from 3-8. They produced 476 and 908 ppb of aflatoxin, respectively. Af-3-(1 and 2) were more toxigenic and produced 770 to 1354 ppb of aflatoxin and sclerotial production was also abundant. These isolates had wrinkled mycelial mat. Isolate Af-4-(1 and 2) also produced abundant sclerotia and wrinkled mycelial mat. These included the highest producer of aflatoxin (1566 ppb). Production of aflatoxin was related to sclerotial production by an isolate and remarkably high correlation (r= 0.96) was observed between these two parameters.

 

Table 1: Categorization of isolates of A. flavus

Group	No. of cultures	Colour of conidial heads	Isolate No.
Af-1   Af-2   Af-3   Af-4	36   21   29   40	Olive green   Green   Olive green   Yellow	Af-1-1 Af-1-2 Af-2-1 Af-2-2 Af-3-1 Af-3-2 Af-4-1 Af-4-2

 

Seed colonization and aflatoxin production:

Seed colonization of sunflower kernels was maximum (94%) with isolate Af-3-2 and was minimum (23.2%) with Af-2-2 (Table 2). However, aflatoxin was produced maximum by isolate Af-4-2 and minimum by isolate Af-1-1 (Table 2). So, there was no correlation in these parameters.

 

Wide variation in the sclerotial production and colour of conidial heads of A. flavus isolates from sunflower is confirmed by the present investigation. These characters were stable within an isolate after repeated sub-culturing over a period of time, which was 10 months in the present investigation. A prominent observation was that conidial heads with colour towards green produced less of aflatoxin compared to heads which had colour towards yellow. Isolates in groups Af-1 and Af-2 produced less a smaller number of sclerotia and had low aflatoxin production as compared to the isolates in the groups of Af-3 and Af-4 which produced more sclerotia and correspondingly, the aflatoxin production was also higher. Also found toxigenic strains^17,19 of A. flavus produced higher number of sclerotia whereas this number was reduced or even nil in case of non-aflatoxigenic strains.

 

Table 2: Cultural Characteristics, Seed Colonization, and aflatoxin production in different isolates of A. flavus

Isolate No.	No. of Sclerotia cm-2	Mean radial growth (cm)	Colony texture	Seed Colonization (%)	Aflatoxin production (ppb)
A f – 1 – 1 A f – 1 – 2 A f  – 2 – 1 A f – 2 – 2 A f – 3 – 1 A f – 3 – 2 A f – 4 – 1 A f – 4 – 2	0 3 3 8 10 19 19 22	10.9 11.2 10.4 10.7 12.0 11.3 11.0 10.6	Slightly wrinkled Plane Plane Plane Wrinkled Wrinkled Highly wrinkled Wrinkled	55.8 55.7 66.7 23.2 76.5 94.0 52.1 76.1	394 666 476 908 770 1354 1120 1566
CD (P = 0.05)	2.6	0.2		6.4	80

 

The results on colonization of sunflower kernels by A. flavus indicate that higher colonization does not necessarily means higher toxic production by the isolate. The results clearly indicate a positive and significant correlation between the two parameters. The significant interrelationship between seed mycoflora and aflatoxin B1 accumulation on oil seeds were also studied¹⁸. From the present findings, it is concluded that quantitative aflatoxin production by an A. flavus isolate is correlated with its cultural characters, viz., colour of conidial heads and sclerotial production and combination of an isolate in vitro. In case of infection of sunflower by A. flavus or even isolation of an isolate from sunflower, it can be possible to evaluate the potential danger the isolate can exhibit in terms of seed colonization and aflatoxin production, by analyzing its cultural characteristics.

 

CONCLUSION:

A total of 126 cultures of A. flavus were obtained from sunflower seeds after the harvest which was obtained from 8 different locations from the districts of Rohilkhand region in Uttar Pradesh. The result of Aflatoxin biosynthesis genomics and cluster variability among A.  flavus population provide a new avenue  in understanding  the mechanisms governing aflatoxin  synthesis  as  well  as pathogenic potential of A. flavus and toxicological risks. Regular monitoring of toxigenic mycoflora, reliable data and sufficient scientific information are pre-requisite to develop effective strategies to identify and control the toxigenic fungi in food and feed. Therefore, the envisaged study used a polyphasic approach for the identification of toxigenic Aspergillus  species involved in  feed ingredient contamination  and  their  aflatoxigenic  gene profiling,  to scrutinize  possible  genetic  pathway  of  aflatoxin production. This will ultimately lead to prevent the human population from the health hazards of aflatoxins and the development of target-specific fungal growth inhibitors.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal, Government Girls Degree College, Bareilly, UP, for providing necessary facilities and infrastructure to carry out the research work. We also extend our sincere thanks to Dr. Md Firoze Quamar, Scientist “D,” Birbal Sahni Institute of Palaeosciences (BSIP), Lucknow, UP, for encouragement and scientific guidance.

 

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Accepted on 28.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):5063-5066.