INTRODUCTION:

Pearl millet (Pennisetum glaucum L.) is a member of the Poaceae family. This millet variety is a substantial commodity in India and Africa and is significantly cultivated. Millets, a fundamental grain in numerous Asian and African meals, have existed for an extended period ¹.

For approximately 10,000 years, eastern India has been growing them. Millets are abundant in a variety of minerals and vitamins, such as folic acid, niacin, B17, B6, magnesium, potassium, and zinc. Pearl millet is a main fare in its native Subcontinent and some African countries. It is prepared in many different kinds of methods, such as cereal, veggies, bread (both made and unleavened), as well as intoxicating beverages. The stalks and foliage are highly valued for their potential applications as fodder for cattle, energy sources, and construction materials ². The widespread spread of plant diseases through seeds has been acknowledged for a long time. Symptoms of these viruses include a poor crop stand, reduced plant development and productivity, and germination-related seed rots and seedling death. Abortion, decay, necrosis, diminished or obliterated germination potential, and damage to seedlings are all potential consequences of pathogens that reside in seeds ³. When the ailment spreads locally or systemically, these issues can progress to later phases of plant development. As a plant disease, fungi are responsible for one of the most critical functions in food deterioration. In addition to reducing crop yield and quality, fungal infections in plants can also pollute cereals with mycotoxin, which are toxic secondary metabolites produced by the fungus. Severe economic losses result from this are involved ⁴.

Mycotoxin are substantially detrimental to the general population when they appear in our food systems and tissues, as they are kidney-damaging, immunotoxic, and mutagenic. Their chronic and acute consequences are susceptible to both animals and humans, and they may manifest as respiration challenges, abnormalities in the nervous system and spinal cord, the heart and lung function, and the intestines among other things ⁵. The possible influence of these toxins in the human liver and esophageal malignancy, a rise in susceptibility to diseases, particularly in youngsters, a decrease in lifespan for people beneath the age of five years, and a decline in pediatric mortality are all major concerns ⁶.

Consequently, the well-being of both people and animals is at risk due to the production of mycotoxin by fungus in grain. The objective of the current study was to find and describe a number of fungal species that were associated with millet and were purchased from a variety of marketplaces in the Rohilkhand region of Uttar Pradesh, India. The investigation was also intended to ascertain the mycotoxin profiles of the cereals.

MATERIALS AND METHODS:

1. Collection of Samples:

The seed samples of pearl millet (Pennisetum glaucum L.) used in this study were obtained from markets in the Rohilkhand region of Uttar Pradesh, India, notably the districts of Bareilly, Pilibhit and Moradabad. Over three months, the millet was acquired from three vendors in each market once a month. Each sample was packed in a sterile nylon bag and delivered to the Phytopathology laboratory of the Department of Botany at the Government Girls Degree College in Bareilly. The temperature and relative humidity levels of the purchased samples were recorded daily and kept in a cold, dry place. The study lasted six months, from January to June of 2023.

2. Sample Preparation and Fungus Isolation:

The dextrose agar medium was poured into a sterile Petri plate. Subsequently, the medium was permitted to consolidate in an atmosphere that was sterile. The millet grains were surface sterilized with a 1.0% solution consisting of sodium hypochlorite (NaClO) and subsequently cleaned three times with sanitized distilled water before being placed on agar on Petri plates. A total of forty grains of millet were collected and transferred to plates with PDA using sterile forceps. The contaminated dishes had been incubated at a temperature of 28-30°C for a duration of 4 to 6 days. The total quantity of colonies observed on the glass plates was recorded. After conducting additional research, each isolate was identified ^7,8.

3. Identification of Fungal Isolates:

The cultural characteristics of the single fungus were carefully documented and monitored. To construct a wet mount on the microscope slide for each isolate, LCB (Lactophenol cotton blue) was employed for staining. When the fungal specimens were examined under the microscope with the X4 and X10 objective lenses, the exact morphological features of each fungal species (isolate) were documented. An authoritative handbook for fungal identification was used to match the isolate features to those mentioned there.⁹

4. Calculating the frequency of fungal isolate occurrence:

To figure out the frequency of each isolate, we counted the number of distinct fungal species present in each sample at every sampling site throughout the research period. The calculation is performed in the following manner:

Frequency = 𝑡/𝑛× 100/1

t= the number of instances of the individual isolate during the specified time.

n= the total quantity of fungal species isolated during the duration of the investigation.

5. Mycotoxin investigation:

The samples were properly mixed, then 20 grammes of each sample were homogenized in an industrial blender for three minutes. Parafilm was placed on top of each combined sample after adding 100 milliliters of 70% methanol. They were shaken for 30 minutes using an orbital shaker set to 400 rpm. The samples were then cleaned through 185mm Whatman's filter sheets. Filtrates were moved to separate funnels, and the leftovers were discarded. The separating funnels' filtrates were each supplemented with 25 mL of dichloromethane and 20 mL of distilled water. The dichloromethane is subsequently mixed with the filtrate and the filtrate-to-water link is dissolved by mildly shaking the separating receptacles and covering them. The separating funnels were settled and separated using conical flasks. Two distinct layers of filtrates have been established. Filtrates were located at the summit of the stack, while toxin extracts were located at the base. The bottom filtrates were transferred to sample containers after passing through pleated filter paper that had been processed using twenty grams of hydrous sodium sulfate. In the strata, additional containers for samples were loaded with toxicity extracts. The sample receptacles were stored in a fume container overnight to allow them to dry. The next day, it was discovered that the cups were dry and empty since the dichloromethane had evaporated, leaving the pollutants inside. The containers were poisoned and one ml of dichloromethane was put in to dissolve it. Before analyzing the materials using thin-layer chromatography with an examining densitometer, they were put into 1.5 ml Eppendorf tubes. All of the values were noted down ^10,11.

RESULTS:

A. Fungal Isolates from Millet Samples in Various Districts of Rohilkhand Region:

Throughout the course of the study, pearl millet samples from the markets in Bareilly, Pilibhit, and Moradabad were used to isolate the following fungi depends on their morphological features.: Aspergillus fumigatus, Rhizopus stolonifer, Curvularia species, Alternaria brassicicola, Fusarium species Mucor mucedo, A. flavus, A. niger, Penicillium species., Nigrospora oryzae, Sporendonema species., and Acladium conspersum were isolated.

B. Frequency percentage of fungus on millets from three major selected locations:

Table 1 displays findings for the frequency percentage of fungal occurrence on the seed’s samples of pearl millet. A. fumigatus (25.5%), Rhizopus stolonifer (14.6%), Curvularia spp. (12.0%) were primarily isolated from samples taken in Bareilly. Rhizopus stolonifer (22.5%) and Mucor mucedo (13.0%) were more commonly isolated from the Pilibhit market. A. niger (35.4%), Rhizopus stolonifer (18.6%) and Aspergillus flavus (15.6%) were also from Moradabad market obtained during the first sampling.

During the second sampling period, samples from three markets showed the following percentage occurrence of fungal frequency: Bareilly market had Aspergillus flavus (35.7%), Aspergillus niger (28.0%); Samples taken from market of Pilibhit had Penicillium (23.9%), Aspergillus flavus (27.5%) and Rhizopus stolonifer (31.2%), while samples collected from the market of Moradabad had A. fumigatus (67.7%), Aspergillus flavus (13.8%) Rhizopus stolonifer (11.8%) and Penicillium spp. (9.7%) whereas Alternaria brassicicola and Fusarium spp. showed the lowest frequency of 1.4%.

A. flavus (71.6%) was more predominant from the isolates collected from Bareilly during the third sampling, over the isolates of A. fumigatus (7.9%) and Alternaria brassicicola (6.7%) respectively. A. niger (32.7%), A. fumigatus (25.5%), and A. flavus (24.6%) were mainly observed from Pilibhit samples whereas samples taken from market of Moradabad A. flavus (55.8%), A. niger (26.9%), A. fumigatus (10.6%) were mainly isolated whereas Nigrospora oryzae (0.6%) showed insignificant frequency percentage occurrence during the third sampling (Table 1).

C. Average frequency (%) of fungus isolated from seeds of Pearl millet samples in three study sites:

The average frequency (%) of fungi in pearl millet differed significantly across distinct regions. The most frequently detected fungal species across all locations were Aspergillus fumigatus, which was present (25.3%), A. flavus (23.8%), and a species of Aspergillus niger (21.6%). Sporendonema spp. and Nigrospora oryzae were the fungi that were observed in isolation the least frequently, with a frequency of 0.4% and 1.2%, respectively. As shown in Table 2.

Table 1: Frequency percentage of occurrence of fungal pathogens on pearl millet from study sites

Sr. No.	Fungal isolates	Locations
		Bareilly			Pilibhit			Moradabad
		L1	L2	L3	L1	L2	L3	L1	L2	L3
1	Aspergillus fumigatus	25.5	15.4	7.9	-	13.6	25.5	-	67.7	10.6
2	Rhizopus stolonifer	14.6	2.4	2.6	22.5	31.2	12.6	18.6	11.8	3.8
3	Fusarium species	9.8	6.6	-	-	13.9	11.2	-	1.4	-
4	Alternaria brassicicola	7.9	-	6.7	-	-	-	-	1.4	2.0
5	Curvularia species	12.0	-	5.8	4.3	2.7	-	-	-	-
6	Mucor mucedo	6.8	5.7	2.4	13.0	-	5.7	7.8	-	3.6
7	Aspergillus flavus	-	35.7	71.6	11.5	27.5	24.6	15.6	13.8	55.8
8	Aspergillus niger	-	28.0	-	-	2.8	32.7	35.4	2.8	26.9
9	Penicillium species	-	6.4	-	-	23.9	-	-	9.7	-
10	Nigrospora oryzae	-	2.5	-	-	-	-	-	-	0.6
11	Sporendonema species	1.9	-	-	-	-	-	-	-	-
12	Acladium conspersum	-	-	-	-	-	-	-	-	-

Key note: L= Locations; - = absent

Table 2: Frequency (%) of occurrence of fungi isolated on seed samples of Pearl millet in three locations:

Sr. No.	Mycoflora	Number (Average frequency %)
1	Aspergillus. flavus	57 (23.8%)
2	A. niger	53 (21.6%)
3	Acladium conspersum	2 (0.7%)
4	Alternaria brassicicola	7 (2.8%)
5	Aspergillus fumigatus	62 (25.3%)
6	Curvularia species	8 (3.3%)
7	Fusarium species	9 (4.3%)
8	Mucor mucedo	6 (2.5%)
9	Nigrospora oryzae	1 (0.4%)
10	Penicillium species	7 (2.8%)
11	Rhizopus stolonifer	15 (5.9%)
12	Sporendonema species	1 (0.4%)

D. The frequency (%) of fungi associated with pearl millet stored for three months in three distinct locations:

When examining the impact of different storage periods on the quantity of fungi on millet from pearls, the two most prevalent species were A. fumigatus (12.5% probability) and R. stolonifer (19.8% probability), while Sporendonema spp. were the least prevalent (0.5%). After two months of preservation, the probability of Acladium conspersum was 3.1%, while that of A. flavus was 53.2%. A. niger was the most prevalent fungus after three months of storage, with an average frequency of 52.7%. Penicillium species followed, with the frequency of only 0.5%. Table 3.

Table 3: The frequency (%) of fungi associated with pearl millet stored for three months in three separate locations

Sr. No.	Mycoflora	Months of storage
Sr. No.	Mycoflora	1 month	2 months	3 months
1	Aspergillus niger	-	17.0	52.7
2	Penicillium spp	-	5.4	0.5
3	Nigrospora oryzae	-	5.7	-
4	Acladium conspersum	-	3.1	-
5	Sporendonema spp.	0.5	-	-
6	Alternaria brassicicola	2.9	-	2.3
7	Fusarium spp	3.5	-	-
8	Curvularia species	6.8	6.1	2.1
9	Mucor mucedo	9.8	5.7	1.9
10	Aspergillus flavus	10.5	53.2	35.8
11	Aspergillus fumigatus	12.5	4.8	3.6
12	Rhizopus stolonifer	19.8	5.6	5.0

E. Mycotoxin Quantification Analysis:

The mycotoxin examination revealed that pearl millet samples contained four aflatoxins: B1, B2, G1, and G2. The most hazardous sample (millet 3) came from the Moradabad market and yielded 886 ppb of aflatoxin B1. The sample from Pilibhit (millet 2) was slightly less toxic at 485 ppb, while the sample from Bareilly market (millet 1) was the least toxic at 231 ppb. The Pilibhit market had a larger concentration of Aflatoxin B2 at 78ppb than the Moradabad market (72ppb) and the Bareilly market (55ppb). In the Bareilly market, aflatoxin G1 was found to be higher (68ppb) than in the Pilibhit market (41ppb). However, in the market of Moradabad, aflatoxin G1 was recorded below the detection limit as zero. In the Bareilly market, Aflatoxin G2 was only detected at 35 ppb. In the Pilibhit and Moradabad marketplaces, the G1 level was less than the detection limit and was registered as 0. The presence of aflatoxin B1 in the pearl millet samples makes them very hazardous. (Table 4).

Table 4: Mycotoxin quantification results:

Sample type	Locations	Sample ID	Mycotoxins: Aflatoxin (ppb)^bc
Sample type	Locations	Sample ID	B1	B2	G1	G2
Pearl Millet	Bareilly	1	231	55	68	35
Pearl Millet	Pilibhit	2	485	78	41	0
Pearl Millet	Moradabad	3	886	72	0	0

Note: a. Recovery of toxin =>85%

b. Zero means the aflatoxin level is below detection limit of the analytical method (1ppb)

c. Values are means of two subsamples of each sample

DISCUSSION:

The research identified twelve distinct varieties of fungus and ten distinct genera in pearl millet grains. Of the total number of fungi, all three were Aspergillus species, including fumigatus, niger, and flavus. The four most prevalent millet fungi are Rhizopus spp., Aspergillus niger, also known as Aspergillus flavus, Mucor mucedo, as well as Aspergillus fumigatus. Each of the five isolates is mold, and the large number of spores and conidia in the air is indicative of their identity. The fungal isolates that were frequently observed in this study are consistent with an earlier report by Vanisha, et al. (2011) ¹² and Hussain, et al. (2009)¹³. Singh and Swami (1994)¹⁴ also purportedly isolated these same fungi¹⁵.

In comparison to other locations, the bulk of fungal species were more common in Bareilly by the second month. For example, the genus Aspergillus has a global range but is more common in tropical and subtropical climates. This observation is consistent with Prescott et al. (2005) ¹⁶. They discovered that A. niger was the most common mould in the natural environment. In the investigation conducted by Mousa, et al. (2015)¹⁷, Aspergillus niger was primarily isolated from all locations and for the majority of the storage months.

Aspergillus and Penicillium Species are the most prominent fungus, also observed by Jurjevic et al. (2007)¹⁸. Certain fungus discovered in this investigation have been associated with health concerns in both human beings and animals. Julie and Jacquline (2000)¹⁹ also demonstrated that the Mucorales, including Rhizopus species., Mucor species, and Sporendonema species, are responsible for the preponderance of human illness.

Numerous unique mold species are associated with the production of mycotoxins^20,21. This is observed in Aspergillosis, which is caused by Aspergillus species. These fungi produce several mycotoxins, included cyclopiazonic acid, roquefortine C,ochratoxin A,verrucosidin, citrinin, penicillic acid, and patulin ²². The findings of Van Egmond and Jewers (1987) ²³ are compatible with the discovery of aflatoxin in millet samples. According to the WHO, AFB1 is the most significant and toxic of all aflatoxins, causing the majority of human and animal disorders. Nevertheless, AFB2 was detected in all of the samples, which was in direct opposition to their report. The WHO (2018)²⁴ report, which asserted that the absence of AFB1 precludes the presence of any other aflatoxins, is also contradicted by the presence of AFG1 and AFG2, as well as other aflatoxins, in the millet grains²⁵.

Aflatoxins induce acute toxicity, immunosuppression, mutagenesis, teratogenicity, and malignancy. The liver is the primary target of carcinogenesis and toxicity. In 1987, the International Agency for Research on Cancer (IARC) ²⁶concluded that naturally occurring aflatoxins had sufficient evidence to suggest carcinogenicity in humans after examining epidemiological and laboratory investigations. According to the World Health Assembly, AFB1 is the most pernicious and potent of the aflatoxins, and it is responsible for the preponderance of maladies in animals as well as humans.

Aflatoxin B1 is a significant issue in the context of food and feed commodities, both during and after harvest. Shanahan et al. (2003)²⁷ have also identified it as a significant hazard to human health. Van Egmond and Jewers ²⁸established the upper limit for aflatoxin in food commodities to be between zero and fifty parts per billion in 1987. Aspergillus species were the most frequently encountered fungal contamination in millet and pearl samples during this investigation. These species are recognized for producing a variety of mycotoxins, besides to being kidney-damaging and cancerous to human beings and other animals^{29, 30, 31}.

Aflatoxin has been associated to carcinogenesis and toxicity in both human and animal populations. Aflatoxin-induced disorders are commonly known as aflatoxicoses. Acute aflatoxicosis leads to death, whereas chronic aflatoxicosis causes immunological suppression, cancer, and other "slow" degenerative disorders. The liver is the primary organ that is affected by the feeding of Aflatoxin B1 to fish, poultry, rodents, and nonhuman primates, resulting in liver injury^.32.

Aflatoxin is permissible in feed cereals and feeds in the United States at a maximum concentration of 20 parts per billion (ppb); however, there is no tolerance for aflatoxin in foods intended for human consumption. Nevertheless, these guidelines were updated.

CONCLUSION AND RECOMMENDATIONS:

This study discovered connections between several kinds of fungus and pearl millet both during preservation and at their point of sale. According to the findings, the fungi Aspergillus flavus and A. niger were the two most common types of fungus. Mycotoxins, which may cause damage to humans and other animals, are a major issue due to the existence of many fungus species linked to the millets used in this research. Effective measures for preventing or considerably reducing fungus contamination and dissemination in millet and its byproducts should be used by farmers from planting to storage. Several methods exist for reducing fungal infections, including as altering the growing sequence, treating the seeds, and using biocontrol. Fertilizer, irrigation, and dense growth of plants are some of the agricultural measures that may be used to reduce mycotoxin.

After implementing the previously described measures, it is essential to carefully manage the storage conditions of millet grains and their derivatives, namely by controlling temperature and moisture levels. Fungi typically flourish in humid or wet conditions; so, it is advisable to keep millet grains in dry surroundings to minimize fungal contamination. It is advisable to avoid storing millet grains for extended periods before disposing of them to prevent the accumulation of pollutants, such as fungi. Incorporating millet grain seed health testing into the national seed quality system is necessary. To ensure that seeds remain viable and capable of germination, the act of planting seeds must be carried out without the presence of any seed-borne diseases. Ultimately, further investigation is necessary to determine the uniformity of mycotoxins and fungi that have been identified from different periods, to establish the frequency and intensity of diseases under favorable circumstances.

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Received on 22.06.2024 Revised on 08.02.2025

Accepted on 31.07.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):91-96.

DOI: 10.52711/0974-360X.2026.00014

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