Identification of mycotoxin producing fungi and conditions leading to aflatoxin contamination of stored foodgrains
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Eugene B. Smalley
A mycological assessment of Thai maize was carried out with DOA specialists. Aspergillus flavus in the air was detected on and around stored maize, in fields near piles of ears, but not in harvested fields. A. flavus contamination was higher in maize weevil (Sitophilus zeamais) infested maize kernels than in noninfested kernels. Weevils carried a significant body burden of A. flavus and Fusarium moniliforme spores. Diploidia zeae (not A. flavus) was the major pathogen attacking Thai maize. F. moniliforme, F. semitectum and Penicillium citrimum were found in all maize samples and may present a greater potential contamination problem than A. flavus. Recommendations include: (1) Mycological analysis should accompany all aflatoxin surveys. (2) Priority should be given to recruitment of an analytical chemist in mycotoxicology. (3) Advanced staff training abroad should be programmed into future UNDP/FAO projects. (4) Develop an in-depth research program on the role of sclerotia, insects and arthropods on the development of aflatoxin in maize. (5) Schedule informed research discussions among international teams cooperating with DOA, and publish results of aflatoxin investigations in international scientific journals.
AFLATOXIN IN MAIZE: CURRENT STATUS AND INTERNATIONAL COOPERATION
Aflatoxin contamination in maize is the cause of great concern in Thailand. This was expressed dramatically at a recent (12119186) symposium on "Aflatoxin in Maize" sponsored by the Thai Maize and Product Traders Association (2). Farmers, traders, silo managers, feed association, Department of Agriculture (DOA), University, Ministry of Agriculture and Ministry of Trade representatives discussed the problem and defined present guidelines for prevention and control. An intense question and answer session followed each speaker. It was apparent that present levels of contamination are unacceptable and that integrated, economic control measures are needed.
Various groups in Thailand produce estimates of aflatoxin contamination in the maize crop (DOA, OMIC, SGS-FAr East Ltd., etc.). Importing countries determine and set standards for aflatoxin concentrations in the incoming maize. Mechanisms for national data collection, collation and assessment of sampling accuracy and analytical methods are not presently available. Divergent estimates of crop contamination and the resultant uncertainty can and are used to manipulate and control maize prices in export trade. Because the DOA has only limited staff, expertise and facilities to address the problem, it has entered into several joint International Cooperative Projects to solve problems and improve the national capability.
Field observation. Farmers field observations were only made on the second crop which was harvested under hot, dry rain-free conditions. As such, these observations and conditions may differ from the situation in the first crop of maize grown and harvested in the rainy season. A high incidence of ear rot was common in all fields examined. The brown dry-rotted ears were very light in weight and cob and kernels offered little resistance to breakage. Frequently in moist ears, white mycelium could be seen massed between the kernels. The incidence of rot in one field we examined near Mauklek involved at least 20% of the crop. Pure cultures of Diploidia zeae (= Stenocarpella maydis) were isolated from all ears exhibiting these symptoms. The identity was confirmed from pycnidia forming in the isolation plates on some of the decayed kernels. We concluded that the variety Suwan 1 was probably extremely susceptible to this pathogen. We also saw evidence of this fungus with more limited rot in association with corn earworm (Heliothis sea.).
Early maize weevil (Sitophilus zeamais) infestations were observed in ears being picked. These infestations were usually at the tips of otherwise sound ears but were rearly extensive. No weevil infestations were observed in good quality second crop shelled kernels on the drying floor, but 3 to 4 month stored first crop kernels were heavily infested at all middleman warehouses visited. In one instance fumigated bags of shelled maize were literally black with killed weevils. Piles of dead weevils 8 to 10 cm deep surrounded the stacks of bags. Upon closer inspection, we found that the dead weevils were yellow-green with conidia of A. flavus. At the silo, maize is regularly migated 2 times in 4 months with CELPHOS (cost 2.5 baht/ton), but it was not an exaggeration to say that in and around the silo builidings and elevators weevils were everywhere and we carried them away on our clothes.
Weevil infestations were heavy in all samples of stored first crop maize we examined with levels ranging from 1% (30/430) in Phetchabun to almost 18% (371248) at Lopburi. Maize at the silo level often had infestations of 20% or more, and it was apparent that these infestations were difficult to estimate when the larval stage is still inside the kernel.
Dilution plate counts from Pakchang first crop maize weevils reveal 133 propagules of A. flavus/ weevil and 576 propagules of Fusarium moniliforme. Maize weevils carried a great collection of other fungi including A. niger, A. glaucus, A. candidus, Penicillium islandicum, P. citrinum, Paecilomyces, Acremonium, Epicoccum, F. semitectum, yeasts and many others.
Air samples at Phetchabun revealed 4.7 propagules A. flavus/10 min. exposure per plate outside the merchants warehouse. Inside the same warehouse A. flavus counts were 4 times greater (19.0 propagules/10 min). A. flavus counts at the hill farm drying shed were 11.0 propagules/plate and increased to 21/plate near the recently shelled cob pile. No A. flavus was detected in the maize fields. The highest A. flavus air counts in the Mauklek area were recorded in a maize field near a workers loading picked ears into bags (16 propagules/10 minj. No A. flavus was detected in the undisturbed maize field after picking. All plates at all locations trapped uncountable numbers of F. moniliforme conidia and the Mauklek plates also trapped additional large numbers of F. semitectum propagules. Maize cobs, stalks and debris apparently were not good hosts for A. flavus survival. No A. flavus was detected in any of the limited number of samples cultured, but moniliforme was omnipresent. Time did not permit a total assessment of A. flavus contamination of all the debris sample collected.
Many different fungi were isolated from surface sterilized maize kernels from the various sources. Forty to 50% of sound first crop kernels from local middlemens warehouses contained A. flavus, but weeviled kernels from the same sources were 100% infected. In contrast no A. flavus could be detected in second crop kernels (without weevil infestation) taken directly from the drying floor, although 30% and quality control of postharvest maize-aflatoxin). The cost of the program (63 million baht = $2.4 million) was shared jointly by Thailand and Japan (1). Aflatoxin survey aspects of this project were reviewed by Nesheim (17).
The project included studies which followed the progress of aflatoxin development throughout the various stages from the field to the middleman's warehouse and finally at the silo. Although experimental designs and statistical validity of comparisons left something to be desired, in most instances the conclusions seemed valid and have been borne out by later work (2,12). Their work indicated that production of aflatoxin begins at harvest and increases in storage until moisture concentrations have been reduced to 15% or less. They concluded that grain precontamination with aflatoxin is transferred directly to the silo company with little further change. They recommended introduction of more effective drying facilities at both farmer and middleman levels, and shifting the crops harvest time to the dry season through the use of delayed planting and short maturing varieties. (Heated discussions on this point took place at the Bangkok Aflatoxin Symposium.) Early crop harvest (4 weeks before maturity) resulted in severe aflatoxin contamination (50 ppb after 20 days storage), but none developed under similar storage conditions in maize picked 2 weeks before maturity (2). Aflatoxin concentrations declined after 20 days storage, and reached levels of 11 ppb in 60 days. No explanation was given for this interesting result.
The Thai-Japanese project also studied the microbiology of fungi in harvested corn from farm through middlemans storage to the silo, Aspergillus flavus was detected 9% of kernels in the farmers field, and had increased to 40% by the time it reached the silo. Fusarium moniliforme was omnipresent in the grain from the farmers field (70% of the kernels) but declined to 21% by the time it reached the silo. The percent of other commonly detected fungi (Penicillium citrinum, P. islandicum, Fusarium semitectum, Botryodiplodia) varied but did not change remarkably between farm and silo. A. flavus, P. citrinum, P. islandicum, F. moniliforme and F. semitectum are all well known mycotoxin, producing fungi (5,15)..
All kernels from the ear rot yield Diploidia zeae. Overall, F. moniliforme and Penicillium citrinum were the second and third most commonly encountered fungi in maize kernels. In these studies F. moniliforme and F. subglutinans could not be distinguished with certainty in the isolation plates and required the preparation of single spore culture on carnation leaf agar (16) for positive identification. For purposes of these preliminary studies both were classified as F. moniliforme (sensu Snyder and Hanson) (16).
In our brief studies on the mycology of Thai maize a number of the fungi we commonly detected have been reported to produce mycotoxins in association with various mycotoxicoses. Aside from Aspergillus flavus (aflatoxin) these included Diploidia zeae (Diploidiosis), F. moniliforme (Equine Leukoencephalomalacea, Esophageal Cancer, Moniliformin), F. subglutinans (Fusaric acid, Moniliformin, Esophageal Cancer), F. semitectum (Degnala resease of water buffalo and cattle, Esophageal Cancer, various Trichothecens and Zearalenone). The various Penicillia and Aspergilli are known to produce a large number of toxins and can always be considered potentially hazardous.
It was apparent that A. flavus was not the major fungal contaminant of the Thai maize. Diploidia zeae was detected in all of the maize fields. In addition it caused losses of 20% or more of harvested ears and although less rotted kernels survived sorting, shelling and cleaning it no doubt becomes a significant comparent of the exported commodity. The heavily infected ears can be removed by on-farm ear sorting but in terms of aflatoxin it presents no hazard since A. flavus was never isolated from Diploidia infected ears.
Neither the Thai-Japanese or Thai-British projects recognized the significant Diploidia contamination. It may have gone unrecognised because the fungus rarely produces pycnidia on isolation media, although we found pycnidia fruiting on old rotted kernels. This pathogen has been reported to product a condition in farm animals called diploidiosis, but the identity of the toxin responsible is unclear.
Fusarium moniliforme (with F. subglutinans) may present the greatest threat of all the maize contaminants. Although it appeared not to cause serious ear rot it could be isolated from most kernels. Both fungi are well known toxin producers and have been associated with several serious diseases (15). While F. semitectum was encountered less frequently, it should not be overlooked because of its well known ability to produce the very toxic trichothecenes including 4-acetoxyscirpeneidol, diacetoxyscirpend, monoacetoxyscirpenol, soirpenetriol and T-2 toxin (15). It has also been reported to produce the fungal estrogen zearalenone. This fungus has been associated with the Degnala Disease and sore leg disease of cattle and water buffalo. These diseases are characterized by swelling of the legs with necrosis and gangrene following ingestion of contaminated rice straw.
A. flavus spores in low concentration were present in the air on and around stored maize; they were also present in the fields near piles of ears but were not generally in the air of the maize field. We did not isolate the fungus from maize stalks or maize debris, but only a few samples were studied. A. flavus contamination was almost non-existent in second crop maize in the field, but quickly built up in storage. This contamination was very much higher in weeviled kernels suggestion an important role may be played by the maize weevil in the aflatoxin intensification process. Weevils were shown to carry significant A. flavus contamination, as well as F. moniliforme and P. islandicum and others. Because of the extremely high maize weevil populations in all stages of maize production from harvest and storage to export, its role needs further study. Its role as an important vector of A. flavus has been reported in North America and elsewhere (6,11).
Mycological analysis should accompany and be companion to aflatoxin determinations. Survey and sampling procedures as recommended by Nesheim (17) for aflatoxin could be utilized for mycological assessment as well. Sampling methods might require modification to take into account the unique and complex marketing systems. It is apparent that the quality of the grain exported depends upon the particular exporter and his buyer. It may not be fair to state that Thailand maize contains 100 ppb aflatoxin when 10 ppb second crop maize ("supergrade") is being sold at premium prices.
Some method of centralized aflatoxin data collection and analysis should be developed in which mycotoxin analytical data from both government and private sector sources can be compiled for general use by the National Aflatoxin Director in DOA, as suggested by Nesheim (17).
Role of Sclerotia, Insects and Arthropods
At its best A. flavus is a weak plant pathogen but is a competitive saprophyte with a large capacity to produce conidia on nutritious substrates. However the factors that allow A. flavus to colonize and produce aflatoxin in maize are not well understood. Sclerotia have been reported to play a major role in producing airborne inoculum, but in our brief survey, we failed to find sclerotia in infected maize or in debris. The large weevil populations with natural A. flavus contamination appear to provide airborne capability and wounds for entry. I recommend the development of in depth research on the role of sclerotia, insects and arthropods on the development of aflatoxin in maize.
Mycotoxin production of Fungi from Maize and Groundnuts
The mycotoxin producing capability of fungi associated with Thai maize is largely unknown. I recommend that studies be initiated to determine the toxicity of culture filtrates from the predominant fungal associates of maize. These should include Diploidia zeae, Fusarium moniliforme, Fusarium subglutinans, fusarium semitectum, Penicillium islandicum, Penicillium citrinum and others. Experimental animals for test feeding might include ducklings, thickens or rats. Toxic strains as indicated in feeding trials should be examined chemically for the production of known mycotoxins or possible undescribed new toxins.
Integration of Projects and Publication of Research
The several cooperative international aflatoxin projects now ongoing at the DOA tend to have overlapping goals. These projects in the past have carried out data processing mainly in their home countries with considerable time before final publication of reports. It might be desirable for the various groups working with DOA to have regularly scheduled research discussions where on-going research could be discussed. A considerable body of information and experience has been accumulated by the DOA on the aflatoxin problem in Thai stored food grain. I encourage publication of this information possibly as a feature article with colored photographs in Plant Disease.
NATIONAL GUIDELINES FOR PREVENTION AND CONTROL OF AFLATOXIN IN MAIZE
These guidelines in the form of large colored posters have been distributed by government organizations and the private sector throughout the maize growing areas of Thailand. Twenty thousand were distributed in 1984 and an additional 50,000 in 1985. The following additional practices are suggested for possible addition to the current guidelines.
The cooperation, advice and gracious hospitality of Mrs. Dara Buangswon, Mr. and Mrs. Prawat TanboonEk, Mrs, Kanjana Bhudhasami and her husband, Dr. Sanchai Tontyaporn, Mr. Noppom Nabheerong, Mrs. Prisna Siriarcha and Mr. and Mrs. Martin Nagler are gratefully acknowledged. The dedicated field collecting and laboratory assistance of Mr. Suparat Kositchoreon, Miss Pattamarat Limpisvastis, and Miss Yubon Nathongpoon were indispensable to our mission. I am also indebted to Mr. Chuwit Sukprakorn, DOA, Chief of Storage Pests Department for confirming the identity of the weevils infesting stored maize in our sample collections. The many thoughtful services and assistance of Mr. Prachob Milindachinta, UNDP was very much appreciated. Last but not least I thank Mrs. Rot Srisaura for her kindness and patience with my large scale use of clean glassware, petri dishes and microscope slides.
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