Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage1

APPLIED MICROBIOLOGY, Mar. 1969, p. 360-365 Copyright 1969 American Society for Microbiology Vol. 17, No. 3 Printed in U.S.A. Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage1 R. E. WELTY AND G. B. LUCAS U.S. Department of Agriculture, Market Quality Research Division, and Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27607 Received for publication 6 January 1969 The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%,; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%c; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0 %; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isolated more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species. Control of the fungi responsible for tobacco being classified as damaged (2) depends upon knowing when these organisms invade the tobacco leaf. Previous work (7) reported 73 % of the fungi isolated from damaged (molded) flue-cured tobacco to be Aspergillus and Penicillium. Another paper (8) reported the genera Alternaria, Cladosporium, Epicoccum, Trichoderma, and Nigrospora collectively composed 89.5 and 65.5% of the fungi isolated from tobacco before and after flue-curing, respectively. These reports indicated that fungi which predominate in damaged tobacco differ from those that predominate in tobacco immediately before and after flue-curing. The purpose of this work was to determine (i) the fungi associated with and the moisture contents of flue-cured tobacco at the time of marketing and (ii) whether the fungi isolated from tobacco damaged in commercial storage were present in tobacco on the warehouse floor. Earlier work (7) in this laboratory reported the fungi isolated from damaged tobacco, but did not determine quantitative differences in fungi isolated from damaged and nondamaged tobacco. Therefore, included in this report are data from samples of damaged and nondamaged stored 1 Paper no. 2760 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. 27607. tobacco purchased from the 1967 crop and analyzed for quantitative and qualitative differences in fungi. MATERIALS AND METHODS Marketed tobacco. During September to December 1967, six tobacco auction warehouses in the Middle Belt and six in the Old Belt of the North Carolina flue-cured tobacco markets were visited, and samples of four or five leaves each were taken from the top or center (or both top and center) of 200-lb (91 kg) piles of tobacco offered for sale. The number of samples from each warehouse varied from 6 to 25. A total of 62 samples came from Middle Belt warehouses and 38 samples were from the Old Belt. Immediately after collection, the samples were put into plastic bags, sealed, and brought back to the laboratory in an ice chest. A cork borer was used to cut fifty 9-mm discs randomly from the leaves. The discs were washed in running tap water for 30 min and cultured, five per dish, on Czapek-Dox agar (Difco) with 6% sodium chloride. The cultures were incubated in the laboratory at room temperature (23 to 28 C). The cultured leaf discs were observed for microbial growth after 6 days and at 2-day intervals up to 12 to 14 days. The fungi growing from the discs were identified as to genus whenever possible in the original petri dishes. If they could not be identified in situ, the fungi were subcultured and stored for later identification. The Aspergillus colonies were identified as to species according to Raper and Fennell (6). The 360

VOL. 17, 1969 FUNGI FROM FLUE-CURED TOBACCO 361 number of isolates of each genus or species were totaled and computations were made of: (i) the percentage of samples yielding the fungus, and (ii) the mean percentage of fungus per sample based on counting the number of discs yielding the fungus, dividing the total by the number of samples yielding the fungus, and converting this mean to percentage. Also, the range in isolation of each fungus was tabulated and recorded as the minimum and maximum percentage each fungus was isolated from those samples yielding the fungus. In many samples, more than one fungus grew from each disc. Stored tobacco. Through the cooperation of a commercial tobacco company, 100 samples were obtained of stored flue-cured tobacco previously purchased on markets in North Carolina, South Carolina, and Virginia between September and December 1967. Samples were rated as damaged or nondamaged by a representative of the company at the time they were taken from hogsheads containing approximately 50 ft3 [950 lb (430 kg)] of stored tobacco. The rating of the samples as damaged or nondamaged was retained by the company until all culturing had been completed. After the samples were collected, they were stored at 3 to 4 C until cultured, which was usually within 7 to 10 days. Fifty leaf discs from each sample were cultured as previously described and fungal identifications were made according to the same procedures. The number and kinds of viable fungus spores or reproductive propagules present on or in each sample were determined by a method similar to that used by Christensen et al. (4) and accepted as standard by the American Association of Cereal Chemists (1) for determining the number and kinds of fungi in flour. A 5-g amount of tobacco was put into 500 ml of a sterile solution of 0.15% agar in a Waring Blendor and comminuted for 1.5 min. Dilutions of this suspension were made, and 1 ml of each dilution was placed in each of two petri dishes. Czapek-Dox agar containing 6% NaCl, melted and cooled to 52 C, was added to each dish; the dish was swirled to distribute the suspension and incubated at room temperature. After 2, 4, and 6 days, the dishes were inverted without removing the covers, and the young colonies were counted and marked under magnification of X6 to X 12. The dishes were then incubated until the colonies could be identified, usually after 12 to 14 days. Results are expressed as the number of colonies per gram of tobacco. Moisture content of the samples was determined by the tobacco company by heating subsamples in a ventilated oven at 100 C for 16 hr (5) and is expressed on a wet-weight basis. RESULTS Marketed tobacco. Fungi isolated from tobacco from the Middle and Old Belt markets comprised 11 genera, including 10 species of Aspergillus (Table 1). A total of 12,344 fungi were isolated, of which 11,477 (93.0%) were Alternaria, Aspergillus, Penicillium, and Cladosporium, 577 (4.7%) were the remaining seven genera, and 290 (2.3 %) were unknown. Aspergillus repens De Bary, A. niger Van Tiegham, A. flavus Link, A. amstelodami (Mangin) Thom & Church, and A. ruber (K.S.&B.) Thom & Church were the predominant species of Aspergillus isolated from Middle Belt tobacco, whereas A. repens and A. niger were the predominant aspergilli from the Old Belt. Ten genera and 11 species of Aspergillus were isolated from 62 samples of tobacco from the Middle Belt; for individual samples, some fungi were isolated from as few as 2% to as many as 100% of the discs. When the number of discs yielding a particular fungus were counted and divided by the number of discs cultured, and when this mean was converted to percentages, Alternaria averaged 40.6% per sample, Penicillium averaged 25.8% per sample, and the 10 species of Aspergillus averaged 19.7% per sample. A. niger and A. repens, the two most common species of Aspergillus, averaged 47.8 and 38.0% per sample, respectively. The 38 samples from the Old Belt markets yielded 11 genera and 8 species of Aspergillus. Here, too, some fungi were isolated from as few as 2% of the cultured discs to as many as 100%. The mean percentage per sample, determined exactly as for Middle Belt samples, was 74.0% for Alternaria, 52.5% for Penicillium, and 16.2% for the eight species of Aspergillus. The mean percentage per sample for the two most common species of Aspergillus was 38.0% for A. repens and 36.2% for A. ruber. When the mean percentage per sample for the predominant fungi are compared between markets, Old Belt tobacco yielded considerably more Alternaria and Penicillium and considerably less Aspergillus niger. The mean percentage per sample for A. repens was equal for samples from both belts. A.flavus, A. ochraceus Wilhelm, and A. amstelodami were isolated from fewer samples from the Old Belt than from the Middle Belt and the mean percentage per sample was also lower for these fungi from the Old Belt. A. ruber and A. tamarii Kita were also isolated from fewer samples of Old Belt tobacco, but the mean percentage per sample for both fungi was more than twice that for these fungi from Middle Belt tobacco. The percentage of samples yielding A. candidus Link was higher for Old Belt tobacco, but the mean percentage per sample was about the same for tobacco from both belts. A. versicolor Tiraboschi and A. restrictus G. Smith were isolated from Middle but not from Old Belt tobacco. Cladosporium was commonly isolated from these samples of marketed tobacco. It was isolated more frequently and had a higher average per sample from Old Belt than from Middle

362 WELTY AND) LUCAS APPL. MICROBIOL. TABLE 1. Marketed tobacco:fungi isolatedfrom 62 and 38 samples of tobacco offeredfor sale in the Middle Belt and Old Belt, respectively, of the flue-cured tobacco market Middle Belte Old Belt6 Fungus isolated Samples Samples yielding Meand Range, yielding Meand Range' fungusc fungus" Alternaria... 98.3 40.6 2-94 100.0 74.0 2-100 Aspergillus A. repens... 100.0 38.0 2-96 97.4 38.0 2-100 A. niger... 96.7 47.8 2-100 76.3 10.6 2-100 A. flavus... 83.9 12.4 2-90 52.6 7.9 2-80 A. amstelodami... 82.3 20.2 2-68 13.1 5.6 2-16 A. ruber... 82.3 16.3 2-80 26.3 36.2 2-100 A. versicolor... 77.4 25.0 2-78 0 0 0 A. ochraceus... 75.8 16.0 2-100 52.6 6.6 2-40 A. tamarị...i.... 75.8 8.0 2-100 15.7 22.6 2-98 A. restrictus... 46.8 8.0 2-56 0 0 0 A. candidus... 16.1 6.2 2-28 34.2 4.0 2-22 Penicillium... 96.7 25.8 2-78 94.7 52.2 2-100 Cladosporium... 72.6 10.4 2-50 86.8 24.2 2-78 Syncephalastrum... 50.0 8.6 2-90 10.5 2.5 2-4 Rhizopus... 29.0 6.6 2-28 39.4 13.4 2-72 Nigrospora... 16.1 6.6 2-18 21.1 3.0 2-8 Mucor... 6.4 8.0 2-8 31.6 3.2 2-10 Epicoccum... 0 0 0 28.9 3.4 2-8 Botrytis... 3.2 2.0 0-2 7.9 3.3 2-4 Fusarium... 4.8 2.0 0-2 7.9 3.3 2-4 Unknown... 20.9 5.2 2-26 68.8 7.2 2-30 a Number of samples, 62; moisture content range, 12.6 to 30.2%; moisture content average, 18%. b Number of samples, 38; moisture content range, 17.4 to 29.7%; moisture content average, 21.6%. c Samples yielding fungus determined by dividing the number of samples in which the fungus occurred by the number of samples for the belt. d The mean percentage per sample was determined by counting the number of discs yielding the fungus, dividing the total by the number of samples yielding the fungus, and converting this mean to a percentage. a The range is the lowest and highest number of discs (converted to a percentage) per sample yielding a particular fungus among the total samples for the belt. Belt tobacco. The remaining seven genera, Syncephalastrum, Rhizopus, Nigrospora, Mucor, Epicoccum, Botrytis, and Fusarium, occurred frequently in some samples but generally did not make up a dominant part of the fungi isolated. The moisture content of the 62 samples from the Middle Belt ranged from 12.6 to 30.2% and averaged 18.0%. Fourteen of these samples were at or below 15.9%, 38 samples were between 16.0 and 19.9%, and 10 samples were at or above 20%. The 38 samples from the Old Belt had higher moisture contents than Middle Belt samples. The range was 17.4 to 29.7% and the average was 21.6%. Eleven samples were at or below 19.9%, 23 samples were between 20.0 and 23.9%, and 4 samples were at or above 24.0%. Although not listed in the tables, the predominant fungus growing from each 50-disc sample was recorded at the end of the incubation period. Alternaria was the dominant fungus in 20 samples from the Old Belt and 5 from the Middle Belt. A. repens was dominant in 15 samples from the Middle Belt and 6 from the Old Belt. Penicillium was almost equally divided between samples from the Old and Middle Belt; it was the dominant fungus in 10 samples from the Old Belt and 7 from the Middle Belt. A. niger was dominant in Middle Belt tobacco 16 times, but was never dominant in Old Belt tobacco. Dominance in the remaining 21 samples was distributed among 5 species of Aspergillus. Stored tobacco. Six genera of fungi, including 8 species of Aspergillus, were isolated from the 74 samples of damaged and 26 samples of nondamaged stored tobacco (Table 2). Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas Alternaria, Cladosporium, Fusarium, and Rhizopus were more frequently isolated from nondamaged tobacco. Damaged tobacco rarely contained

VOL. 17, 1969 FUNGI FROM FLUE-CURED TOBACCO 363 TABLE 2. Stored tobacco; qualitative determination of the fungi growing from 74 samples of damaged and 26 samples of nondamaged tobacco based on culturing 50 discs of tobacco for each sample Damaged" Nondamaged' Fungus isolated Samples Samples yielding Meand Range' yielding Meand Range, fungus" fungus0 Aspergillus repens.. 79.7 53.6 0-100 100.0 50.0 0-98 A. niger... 77.0 45.4 0-100 96.2 31.0 0-100 A. ruber... 71.6 35.8 0-100 96.2 33.0 0-90 A. ochraceus... 67.6 25.2 0-100 57.7 5.6 0-46 A. amstelodami... 36.5 28.4 0-98 69.2 6.4 0-24 A. flavus... 32.4 12.4 0-70 23.1 6.0 0-22 A. tamarii... 23.0 8.6 0-82 11.5 6.0 0-14 A. versicolor... 16.2 25.6 0-100 61.5 5.4 0-12 Penicillium... 81.1 68.6 0-100 96.2 33.6 0-100 Cladosporium... 9.5 3.2 0-4 42.3 5.0 0-14 Rhizopus... 5.4 3.0 0-6 11.5 16.6 0-26 Alternaria...... 1.4 2.0 0-2 96.2 31.0 0-88 Fusarium... 1.4 2.0 0-2 7.7 15.0 0-24 Unknown... 4.1 3.4 0-6 69.2 6.0 0-20 Number of samples, 74; moisture content range, 17.4 to 44.8%; moisture content average, 25.3%; samples yielding 1 to 6 different fungi, 77%; samples yielding 7 to 12 different fungi, 23%. b Number of samples, 26; moisture content range, 13 to 22.2%; moisture content average, 18.3%; samples yielding 1 to 6 different fungi, 11.5%; samples yielding 7 to 12 different fungi, 88.5%. c See footnote c, Table 1. d See footnote d, Table 1. e See footnote e, Table 1. Alternaria and Cladosporium; Alternaria was isolated from only one sample, and Cladosporium was isolated from seven. In any one sample, there were never more than 4% of the discs yielding either or both of these fungi. In contrast, Alternaria and Cladosporium were frequently isolated, 96.2 and 42.3%, respectively, from the 26 samples of nondamaged tobacco. In one sample, Alternaria grew from 88% of the discs cultured. More samples of damaged tobacco contained A. ochraceus, A. flavus, and A. tamarii than nondamaged samples, whereas the reverse was true with A. niger, A. amstelodami, and A. versicolor. The range and the mean percentage per sample of A. repens, A. ruber, and A. niger from damaged and nondamaged tobacco were about the same. However, 96 to 100% of the nondamaged samples yielded these three species, whereas they were isolated from about 70 to 80% of the samples of the damaged tobacco. The mean percentage per sample of A. ochraceus, A. amstelodami, A.flavus, A. tamarii, and A. versicolor was considerably higher for damaged than for nondamaged tobacco; for A. ochraceus and A. versicolor, the difference was fivefold, for A. amstelodami fourfold, and for A. flavus twofold. Moisture content of the tobacco from damaged samples ranged from 17.4 to 44.8%, whereas the range for nondamaged samples was considerably lower, 13.0 to 22.2%. The average moisture content of damaged samples was 25.3 % compared to 18.3% for nondamaged. Most of the samples of nondamaged tobacco (88.5%) yielded from 7 to 12 different fungi, whereas 77.0% of the samples of damaged tobacco yielded only 1 to 6, most of which were Aspergillus species (Table 2). This reduction in the number of different fungi isolated from damaged tobacco was probably due to competition among the fungi during storage, although this hypothesis was not tested. It should be kept in mind that the results from this culture method are of a qualitative nature (i.e., presence or absence of a fungus) and do not reflect a quantitative measure of the fungi present. The tests to determine whether quantitative differences existed for the fungi present on or in damaged and nondamaged samples disclosed that populations of Aspergillus species and Penicillium were higher in damaged samples. Not only were these fungi isolated more frequently from damaged samples, but the relative amounts as determined by mean number of colonies per gram were many times greater for damaged samples than for nondamaged samples (Table 3).

364 WELTY AND LUCAS APPL. MICROBIOL. TABLE 3. Quantitative determination of the number and kinds offungi isolatedfrom 74 samples of damaged and 26 samples of nondamaged flue-cured tobacco obtained from a commercial tobacco companya Damaged Nondamaged Fungus isolated Samples Sanles yeing Mean fungi/g urange of fungi/gd yielging fungi/ge fungi/gd fungus fungus Aspergillus repens... 81.1 12,920,000 0-120,000,000 7.7 22 0-40 A. ruber... 41.9 12,855,000 0-5,000,000 3.8 5 0-5 A. amstelodami... 20.3 1,557,000 0-13,000,000 0 0 0 A. niger... 18.9 9,680,000 0-120,000,000 0 0 0 A. flavus... 5.4 185,000 0-200,000 0 0 0 A. ochraceus... 4.1 900,000 0-2,000,000 3.8 400 0-400 A. tamarii... 2.7 760,000 0-1,500,000 0 0 0 Penicillium... 28.4 10,720,000 0-100,000,000 3.8 5 0-5 a Fungus counts are based on a dilution series of 5 g of tobacco per sample. b Determined by dividing the number of samples in which the fungus occurred by the number of samples for each tobacco class. c The mean number of colonies of a fungus per gram was determined by counting the number of colonies of the fungus for each sample and dividing the total by the number of samples yielding the fungus for each tobacco class. d The range in fungi per gram indicates the lowest and highest number of colonies observed for each fungus among the total samples tested for each tobacco class. A. repens was isolated from 81.1 % of the damaged samples; it had the highest average number of colonies per gram (12.9 million) and had one of the widest ranges in number of fungi per gram per sample (0 to 120 million). The other fungi most frequently isolated from damaged tobacco were A. ruber, A. niger, and Penicillium. Although these three fungi were not isolated from as many damaged samples as A. repens, their relative amounts were large, about 10 to 13 million propagules per g. The other three aspergilli isolated from the damaged tobacco, A. flavus, A. ochraceus, and A. tamarii, were not frequently isolated; when recovered in petri dishes, their populations were lower than Penicillium and the other four species of Aspergillus İn this quantitative test, the number and kinds of fungi present in samples of nondamaged tobacco were relatively small when compared to the fungus population present in damaged tobacco. A. ruber, A. ochraceus, and Penicillium were each isolated from only one sample, and A. repens was isolated from two samples. The four other species of Aspergillus were not isolated. It is apparent from these results that Penicillium and several species of Aspergillus occur more frequently and in higher populations in damaged tobacco than in nondamaged tobacco. The reason this appears to contradict the findings in the qualitative test (Table 2) is that the dilution factor in the quantitative test eliminates low populations of the fungi. Whereas the qualitative test allows a small population (e. g., one spore) to grow and its presence to be recorded, the quantitative test eliminates small populations and gives a more accurate indication of the fungi in the tobacco sample. DISCUSSION The results here reported suggest that many genera and species of fungi can be isolated from marketed flue-cured tobacco. It appears that differences occur in the kinds and numbers of fungi isolated from tobacco sold on different markets, i.e., Old Belt tobacco contained a higher average amount of Alternaria, A. ruber, and Penicillium and Middle Belt tobacco contained large numbers of A. niger, A. amstelodami, A. versicolor, and A. restrictus. Although data are insufficient to attempt a correlation between the number and kinds of fungi isolated and the moisture content of the samples, moisture content might account for these differences. The average moisture content of the tobacco from the Old Belt was higher by 3.6% than the Middle Belt (21.6% versus 18.0%). The role of moisture content in the development and increase of fungi has not been demonstrated for tobacco, but it is well established that moisture content determines which fungus predominates in stored cereal seed (3) Ḋamaged stored tobacco contains a consistently higher average number of Aspergillus species and Penicillium and a consistently lower average number of Alternaria, Cladosporium, Rhizopus, and Fusarium than nondamaged stored tobacco. This result was consistent for both the qualitative

VOL. 17, 1969 FUNGI FROM FLUE-CURED TOBACCO 365 (Table 2) and quantitative (Table 3) tests done with the 100 samples of stored tobacco. Again, these differences in fungi might be due to differences in sample moisture content. The results indicate that marketed tobacco has a fungal flora similar to the fungi isolated from tobacco after flue-curing on the farm (8) and to the fungi isolated from tobacco damaged during storage (7). From this work and previous reports, it appears that a fungal transition occurs whereby the fungi that invade in the field do not increase in storage but eventually are reduced or eliminated by the fungi able to grow in storage. As both field-invading and storage-developing fungi were isolated from nondamaged tobacco sold in warehouses, apparently the fungi isolated from damaged stored tobacco had invaded the tobacco prior to marketing. Earlier reports (7, 8) found Aspergillus and Penicillium species to be frequently associated with damaged tobacco, whereas fungi in the genera Alternaria, Cladosporium, Epicoccum, Trichoderma, and Nigrospora were commonly associated with tobacco on the farm. These reports and the present results correspond to what Christensen and Kaufmann (3) reported for the fungi isolated from cereal grains. Their nomenclature regarding "storage" and "field" fungi are suggested here for future reference to fungi isolated from tobacco. Field fungi, represented by Alternaria, Cladosporium, Epicoccum, and Trichoderma, invade tobacco prior to harvest, and storage fungi, mainly species of Aspergillus and Penicillium, invade tobacco after harvest. How moisture contents affect fungal growth and the role these organisms play in tobacco deterioration are presently being investigated. ACKNOWLEDGMENTS This investigation was supported by Agricultural Research Service, U.S. Department of Agriculture, Cooperative Agreement no. 12-14-100-9088(51), administered by the Market Quality Research Division, Hyattsville, Md. 20782. The technical assistance of Virginia Perez and James T. Fletcher is gratefully acknowledged. LITERATURE CITED 1. American Association of Cereal Chemists. 1962. Cereal laboratory methods, 7th ed. Am. Assoc. Cereal Chemists, St. Paul, Minn. 2. Anonymous. 1965. Official standard grades for flue-cured tobacco. U.S. Department of Agriculture Consumer and Marketing Service, Tobacco Division, Washington, D.C. 3. Christensen, C. M., and H. H. Kaufmann. 1965. Deterioration of stored grains by fungi. Ann. Rev. Phytopathol. 3:69-84. 4. Christensen, C. M., H. A. Fanse, G. H. Nelson, F. Bates, and C. J. Mirocha. 1967. Microflora of black and red pepper. Appl. Microbiol. 15:622-626. 5. Iles, W. G., and C. F. Sharman. 1949. The effect of ventilation in moisture testing ovens. J. Soc. Chem. Ind. 68:174-175. 6. Raper, K. B., and D. I. Fennell. 1965. The genus Aspergillus. The Williams & Wilkins Co., Baltimore. 7. Welty, R. E., and G. B. Lucas. 1968. Fungi isolated from damaged flue-cured tobacco. Appl. Microbiol. 16:851-854. 8. Welty, R. E., G. B. Lucas, J. T. Fletcher, and H. Yang. 1968. Fungi isolated from tobacco leaves and brown-spot lesions before and after flue-curing. Appl. Microbiol. 16:1309-1313. Downloaded from http://aem.asm.org/ on October 2, 2018 by guest