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Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China
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Agricultural Sciences in China 2007, 6(6): 682-687
ScienceDirect
June 2007
Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China DUAN Can-Xing, WANG Xiao-ming, ZHU Zhen-dong and WU Xao-fei Institute of Crop Sciences, Chinese Academy of Agricultural Sciencesflhe National Key Facilities f o r Crop Gene Resources and Genetic Improvement (NFCRI), Beijing 100081, P.R.China
Abstract There is a primary understanding of the dominant fungi in wheat seeds conserved in the National Crop Genebank of China (NCGB) and an evaluation of the healthy status of wheat germplasm propagated in different regions. A total of 1465 wheat accessions were detected for seedbone fungi by blotter, agar plate, and wash tests. By blotter test, 17 genera of fungi, including more than 30 species, were detected in 712 wheat accessions from Shaanxi, Hebei, and Qinghai provinces, China. Alternaria was the most frequently detected in wheat seeds from Shaanxi Province, followed by Rhizopus, Penicillium, Aspergillus, Bipolaris, Cladosporium, Gonatobotrys, Chaetomium, and others. Seedborne fungi in wheat seeds from Hebei Province, with relatively high incidence were Alternaria, Rhizopus, Penicillium, Aspergillus, Bipolaris, Cladosporium, and Fusarium. In the seeds from Qinghai Province, Alternaria, Rhizopus, Bipolaris, Cladosporium, and Trichothecium are important seed-borne fungi. The seed germination was reduced substantially when seeds were infected by Fusarium verticillioides (syn. F. moniliforme), Bipolaris nodulosa, and Cladosporium herbarum. Eighteen genera and 25 species of fungi were identified in 353 accessions from Shaanxi Province using the agar plate test. The dominant fungi were Alternaria, Aspergillus, Bipolaris, Gonatobotrys, Penicillium, and Fusarium. The smut fungi, Ustilago tritici, was detected by the wash test in 400 accessions, but it was low in incidence in 300 seed samples from Shaanxi Province (1.3%), and in 100 samples from Hebei Province (2.0%). Totally 19 genera of fungi were detected in wheat seed samples, and some seedborne fungi were saprophytic and others were biotrophic which could cause seedborne diseases in the field.
Key words: wheat germplasm, seedborne fungi, seed health, detection
INTRODUCTION Plant germplasm resources are viewed as the root and foundation for crop production and breeding. They are also indispensably the strategic wealth for survival and development of human. To date, the crop Genebank system has been established in China and 350 000 crop accessions have been conserved in the long-term Genebank and 280000 germplasms in the medium-term
Genebank. The promotion of crop yield and quality greatly depends on the quality of seeds. Seed health has been considered as an attribute of high quality and one of the most important premises for safe conservation. Seedborne fungal, bacterial, and viral pathogens have some deleterious effects on seed, such as, reducing seed viability, vigor and germination capability, shortening longevity of conservation, and causing physiological changes. Furthermore, some seedborne pathogens are also seed-transmitted, which
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence WANG Xiao-ming, Professor, E-mail: [email protected]
02007,CAAS. All @hts resewed. Published by ElsevierLtd.
Testing of Seedborne Fungi in Wheat Germolasrn Conserved in the National Crop Genebank of China
can cause severe diseases in the field after seed movement at a long distance (Neergaard 1979). Wheat is one of the most significantly staple crops in the world. High quality seeds are important to maintain production, improve yield and quality, and conserve germplasm safely. In recent years, the studies on seed health testing have been increasingly deployed in rice, maize, tobacco, fruit, common bean, annual flowers, American ginseng, leguminousplants, and other crops in China (Liu et al. 2000; Li and Hu 2001; Li et al. 2001; Tan et al. 2002; Yang et al. 2002; Zhang G Z and Zhang X F 2002; Wang and Tylkowska 2003; Wu et al. 2003; Wu et al. 2004). However, the health status of wheat seeds conserved in Genebank remains elusive. In this study, three routine techniques of seed health testing are applied to monitor and analyze the fungi that infect wheat accessions conserved in the National Crop Genebank of China. The aim is to raise a theoretic foundation to control seedborne fungi, to improve seed quality, and to prolong the period of conservation.
MATERIALS AND METHODS Experimental materials One thousand four hundred and sixty-five wheat accessions selected randomly from the National Mediumterm Crop Genebank of China, which were propagated in Shaanxi, Hebei, and Qinghai provinces respectively, were provided by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.
Experimental methods Blotter test According to Wang and Tylkowska (2003) and Mew and Misra (1994), three layers of 9.0 cmdiameter white blotter papers soaked in sterile distilled water were placed in petri plates allowing NUV light to penetrate. Twenty-five wheat seeds were placed in each plate and four plates of seeds per germplasm were tested. The seeds were incubated for 7-10 d at 22°C with alternate 12 h periods of NUV light and darkness. The plates were approximately 40 cm below the lights. The seeds were examined under a microscope and the
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fungi were recorded. Affected seeds rate = Number of seeds infected with some fungimotal tested seeds x 100% Affected germplasm rate = Number of samples infected with some fungi/Total tested accessions x 100% Affected range is the percentage that all accessions infected by the same fungus varied to a certain extent. Agar plate test Wheat seeds for testing were pretreated with 1.O% (v/v) sodium hypochlorite for 2 min and then rinsed thrice with sterile distilled water. The sterilized seeds were placed in a 9.0 cm-diameter PDA plate (potato 200 g, glucose 20 g, agar 20 g, distilled water 1 000 mL). A total of three plates (10 seeds per plate) were tested. Seeds were incubated at 25°C for 7- 10 d to allow fungal growth and sporulation with an 8-h NUV light per day (McGee 1994; Mew and Misra 1994; Michail et al. 1999; Li et al. 2001). Wash test Fifty wheat seeds were added to 10 mL sterile distilled water in a 100 mL flask. One drop of sterile Tween 20 was added to the flask and then the flask was vigorously shaken for 10 min. The suspension was transferred to a 10 mL tube and centrifuged at 3 000 r/min for 5 min. The pallets were resuspended in 1 mL Shear’s solution and examined for Tilletia spp. and Ustilago spp. with the help of a microscope (McGee 1988; Mathur and Cunfer 1993).
RESULTS Wheat seedborne fungi tested by blotter test Seven hundred and twelve wheat samples, obtained from Shaanxi, Hebei, and Qinghai provinces were tested and 17 genera of fungi, including more than 30 species were detected. There were 17 genera of fungi comprising of 34 species infecting wheat seeds from Shaanxi Province, 22 species of fungi derived from 14 genera affecting wheat from Hebei Province, and 10 genera of fungi, including 13 species found in wheat seeds from Qinghai Province (Table 1). Results revealed that Altemaria tenuis was the grain-infecting fungi detected most frequently in wheat seeds, followed by Rhizopus stolonifer, Penicillium spp., Alternaria triticina, Altemaria tenuissima, Chaetomium spp., and others with infection percentages of 5 1.00, 21 .OO, 6.00, 2.80,
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DUAN Can-Xing et al.
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1.90, and 1.90,respectively. There were 0.002%seeds affected by Fusarium poae. A. tenuis was most frequently detected among the 7 12 wheat accessions, followed by R. stolonifer, Penicillium spp., A . triticina, Bipolaris nodulosa, A. tenuissima, and Aspergillusflavus with the percentages being 90.20, 45.14, 43.20, 36.00, 26.13,24.11,and 20.80,respectively, whereas, incidence of Stemphylium sp. and E poae were both 0.14%. Affected range by A . tenuis, R . stolonifer. A. jlavus, Monilia tetrasperma, and Penicillium spp. varied to an extent of 1.0-100, 1.0-100, 1.0-89.0,1.0-85.0,and 1.082.0%,respectively. Therefore, it was confirmed that wheat germplasm was susceptible to some fungi, such as Alternaria, Rhizopus, Penicillium, Bipolaris, and Cladosporium. A, f l a w s was carcinogenic and was poisonous for humans and cattle. Germinability of seeds, infected with some seedborne fungi, such as Fusarium verricillioides (syn. F. moniliforme), B. nodulosa, and Cladosporium herbarum, declined significantly and therefore many seeds even lost viability completely. Therefore, it was crucial to control the
fungi for safe conservation of wheat germplasm.
Analysis of health status of wheat accessions from different regions by blotter test Analysis of fungi infecting 712 wheat accessions from different areas, including 28 accessions from Qinghai. 166 from Hebei, and 5 18 from Shaanxi, indicated that the number of fungi species affecting seeds from Shaanxi markedly exceeded those from Qinghai and Hebei (Table 2), which were probably related to the accessions origin and number of samples tested. A. tenuis, R. stolonifer, and Penicillium spp. were frequently found in wheat seeds from Shaanxi, with percentages of 48.9, 23.9, and 7.53 and the affected accessions rates were 87.2, 55.1, and 54.9% in these three species of fungi, respectively. As a whole, wheat accessions from Shaanxi were subject to invasion from eight genera of fungi, comprising of Alternaria, R h i z o p us , Pen i c i 1 1 i M m , A s p e rg i 11u s , B i p o 1 a ris , Cladosporium, Gonatobotlys, and Chaetomium.
Table 1 Incidence of seedborne fungi detected in 712 wheat accessions by the blotter test Fungus
___.__~
Alrernariir rriricinu .4. renuissirnu .4. renuis Asper,qilh niger 4. f l a w s A . cundidu>
Aspergillus sp. Chaeromiurn spp C. Perithecium C. herharum C. macrocarpum C. oxysporium Dictyoarthrinium quudratum Bipolaris hicolor B. cynodonris B. noduloso B. teframrrii Epicoc cum purpurast'ens Fusarium graminearurn F. rnoniliformc F. pour F. remrrccrurn Gonatohorn s africariu Morrilia rerruspernra Nigrosporu cp Penicilliuni spp. Phomo spp Hhizopus srolonifer Stemphylium sp. Trichotlrecrum roseuni Verticilium lateritium
Affected seeds ('h)
Affected accession (%)
2.80 2.00
36.00 26.13 90.20 14.32 20.80 3.87 11.50 9.18 1.29 7.60 3.30 14.80 0.57 12.60 10.52 24.11
51.00 0.57
1.40 0.10 0.68 1.90 0.02 0.17 0.10 0.40 0.01 0.37 0.22 0.70 0.04 0.02 0.02 0.10 0.002 0.04 1.10 0.81 0.03 6.00 0.01 2 I .oo 0.02 0.58 0.03
2.30 1.oo 1.oo 2.58 0.14 2.44 13.63 5.16 I .72 43.20 0.43 45.14 0.14 9.61 I .29
Affected range - in seeds (Yo) 1.00-61.00 1.00-73.00 1.00-100.00 1.00-49.00 1.00-89.00 1.00-4.00 I .oo-22.00 1 .OO-79.00 I .OO-6.00 1.00- 11.00 2.00-18.00 1.00-20.00 1.00-1.00 1 .OO-8.00 1.OO-6.70 I .OO-14.00 1 .oo-3.00 1.OO-4.00 1 .oo-2.00 1.OO-15.00 1.00-1.00 1.00-4.00 1.00-38.00 1.OO-85 .OO I .OO-3 .OO 1.00-82.00 1 .OO-3.00 1.00-100.00 3.00-3.00 1.00-33.00 1.00-6.00
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Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China
The ratios of seeds affected by A . tenuis, R . stolonifer, and A. triticina from Hebei were 67.9, 11.8, and 1.96%, respectively, whereas, the percentages of accessions infected by A . tenuis, A. triticina, and B. nodulosa reached 99.4,44.0, and 33.1%, respectively. There were seven genera of fungi with high incidence, in c ludi n g A 1 t e rna ri a , R h izop u s , Pen ic i 11 i um , Aspergillus, Bipolaris, Cladosporium, and Fusarium. As far as wheat accessions from Qinghai, some seedborne fungi such as Altemaria, Rhizopus, Bipolaris, Cladosporium, and Trichothecium were prevalent. Wheat seeds infected by A. tenuis were most frequent, followed by A . tenuissima and R. stolonifer with the percentages of 79.3, 6.25, and 4.71, respectively, A. tenuis, A. tenuissima, and B. bicolor were detected frequently in accessions with the percentages of 100,95.7, and 78.3, respectively.
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were discovered in 353 wheat accessions from Shaanxi by using the agar plate test (Table 3). Seedborne fungi, which were frequently detected were A . tenuis, R. stolonifer, A . triticina, and A. tenuissima. The percentages of seeds affected by them were 65.3, 33.2, 11.O, and 10.6 and the accessions infected reached up to 99.4, 67.7, 55.8, and 61.2%, respectively. Affected range in accessions by fungi, such as A . tenuis, R. stolonifer, A. triticina, A. tenuissima, and Gonatobotrys africana vaned to a great extent. Six genera of fungi, including Altemaria, Rhizopus, Aspergillus, Bipolaris, Gonatobotrys, and Penicillium were readily detected by the agar plate test. In addition, Pestalotia macrotricha, which was never reported as a seedborne fungus in wheat seeds, was detected with 0.57% of incidence in the accessions tested.
Seedborne fungi examined by wash test Seedborne fungi detected by arga plate test Eighteen genera of fungi, comprising of 25 species,
A hundred wheat accessions from Hebei and 300 from Shaanxi were tested by wash test for Tilletia spp. and
Table 2 Incidence of seedborne fungi detected in wheat accessions from three provinces by the blotter test Shaanxi Province A. triticina A. renuissima A. tenuis A. niger A. f l a w s A. candidus Aspergillus sp. Chaetomium spp C. Perithecium C. herbarum C. macrocarpum C. oxysporium D. quadratum B. bicolor B. cynodontis B. nodulosa B. retramera E. purpurascens F. graminearum F. moniliforme F. poae F. semitectum G. africana M . tetrasperma Nigrospora sp. Penicillium spp. Phoma spp. R. stolonifer Stemphylium sp. T. roseum V. lateritium
3.16 2.13 48.92 0.65 1.80 0.13 0.83 2.44 0.03 0.19 0.12 0.47 0.01 0.30 0.16 0.70 0.04 0.02 0.02 0.12 0.002 0.03 1.42 0.84 0.04 7.53 0.01 23.90 0.01 0.72 0.04
Affected seeds (%) Hebei Qinghai Province Province 1.96 1.07 67.90 0.36 0.02
1.58 6.25 79.32
0.06
0.50
0.07 0.02 0.12 0.01 0.58 0.54 0.94 0.03
0.67 0.75 2.00
0.25 0.04
0.01 0.10 0.01 0.89
0.29
0.35
0.29
11.83
4.71
0.01 0.02
0.50
Affected accession (%) Shaanxi Hebei Qinghai Province Province Province 33.70 28.40 87.22 18.03 27.30 5.16 14.20 12.22 1.72 8.99 4.21 17.01 0.57 10.10 8.22 21.60 2.49 1.14 1.15 3.25 0.19 2.10 17.83 5.93 2.29 54.92 0.57 55.10 0.19 12.20 1.53
44.00 12.70 99.43 4.22 1.81
39.12 95.70 100.00
10.80
30.42
3.01 0.60 6.02 0.60 16.92 18.10 33.10 1.81
34.81 56.50 78.30
17.40 4.35 0.60 3.61 0.60 3.01
26.12
8.43
21.70
13.92
43.50
0.60 0.60
34.90
Affected range in seeds (76) Shaanxi Hebei Qinghai Province Province Province 1.00-61.00 1.OO-73.00 1.00-100 1.00-49.00 1.OO-89.00 1.00-4.00 1.oo-22.00 1.00-79.00 1.00-6.00 1.oo-11.oo 2.00- 18.00 1.00-20.00 1.00 1.00-8.00 1.00-6.70 1.OO-14.00 1.00-3.00 1.00-4.00 1.oo-2.00 1.00-15.00 1.00 1.00-4.00 1.00-38.00 1.00-85.00 1.00-3.00 1.00-82.00 1.00-3.00 1.00-100 3.00 1.OO-33.00 1.OO-6.00
1.00-16.00 1.00-20.00 4.00-100 1.oo-20.00 1.oo
2.00-6.00 3.00- 13.00 70.00-90.00
2.00
1.00-2.00
2.00-4.00 2.00 2.00-4.00 1.00 1.30-10.00 2.00-8.00 1.00-6.00 2.00
1.00-3.00 1.00-3.00 1.00-5.00
1.oo-2.00 1.oo 2.00 2.00-4.00 2.00 9.00-30.00
1 .oo-2.00
2.00-10.00
1.00-2.00
2.00-100
2.00-4.0 1
2.00 2.00
1.00-3.00
-,the fungus was not found.
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Table 3 Incidence of seedborne fungi detected in 353 wheat accessions by agar plate test Fungus
______~-. .___
A. triticina A . tenuissimo A. tenuis A. niger A. Javrrs A. candidus Aspergillu.5 sp. Chaetomium spp. C. lierbar~ni B. noduloso B. tetramerii E. purpurascens F . moniliforme F..semitecrum G. qfricano M rerraspermn N ig rosporo \p . Penicillium \pp. Pesralotia macrorrichu Phoma spp R. rrolonifer Sremphylinm sp. 7 rnseum
Affected seeds (8) I 1 .oo 10.63 65.32 0.59 0.28 0.01 0.1 I 0.05 0.59 I .93 0.05 0.05 0.03 0.18 3.70 0.09 1 .O1 1.88 0.02 0.03 33.20 0.25 0.5 1
Ustilago spp. There were six accessions with Ustilago tritici spores in 400 accessions, and the incidence of Ustilago tritici was 1.30% in 300 accessions from Shaanxi and 2.0070 in 100 accessions from Hebei. Tilletia spp. was not detected in 400 wheat accessions.
DISCUSSION Most samples uere infected by several species of fungi in 1465 wheat accessions tested. The seedborne fungi could damage seeds at a different degree, causing seed shrinkage or color change. Some fungi, such as Alternaria, Aspergillus, Bipolaris, and Fusarium produced different fungal toxins, which could make changes in the chemical ingredients inside the seeds, reduce nutritive value and viability of seeds, and even cause seed death. In addition, some seedborne fungi were also the causal agents of diseases invading the roots, stems, and leaves of wheat. For instance, A . triticina could cause leaf blight and root rot, F. poae and F. verticillioides led to foot rot and scab, Bipolaris spp. caused spot blotch. and Cladosporium rendered blach head mold (Wiese 1987; Lu 2001). The diseases transmitted by seeds resulted in an increase of difficulty in disease control and in the decline of yield and
Affected accession (8) 55.82 61.21 99.40 13.93 7.37 0.28 2.83 1.42 13.02 21.50 1.13 I .42 0.57 5.38 14.70 0.28 9.63 32.63 0.57 0.85 67.72 5.67 6.80
Affected range in seeds (8) 3.33-86.73 3.33-70.00 10.0-96.73 3.33-10.00 3.33-6.73 3.33 3.33-6.73 3.33 3.33-13.33 3.33-33.33 3.33-6.73 3.33 3.33-6.73 3.33 3.33-80.00 30.00 3.33-26.73 3.33-23.33 3.33 3.33 6.70-96.73 3.30-6.73 3.30-23.33
quality. Seed health testing was deployed widely in America, Europe, and some developed countries, and molecular biological techniques were used to detect seedborne pathogens swiftly. In addition to blotter, agar plate, and wash tests, a fluorescent test was also a routine method and its targeted fungus was Septoria nodorum. At present, the blotter test is most widely applied to detect seedborne fungi. Compared with the agar plate, the blotter test was simpler, more economic, and effective in that certain species of fungi were difficult to identify due to hard sporulation. Certainly, there were some advantages of the agar plate test, for example, the tester familiar with cultural characters of the fungal colony could quickly identify the species of the fungus with a naked eye. In China, crops were subject to infection by many pathogens because they were grown in wide areas and different agricultural ecological regions, and thereby some pathogens, of which some can cause hazardous diseases, were transmitted and diffused by infected seeds. The seedborne pathogenic fungi kept long-term survival in seeds, when the seeds were conserved in the Genebank, under conditions of low temperature and relative humidity, which had a harmful effect on germplasm viability and genetic integrity, and also en-
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Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China
gendered a potential threat to agricultural production when these germplasm was planted and propagated after decades. Therefore, seed health testing could provide a scientific foundation for controlling seedborne pathogens in crop propagates, conservation of germplasm in the Genebank, and monitoring the seed’s vigor in a long-term conservation.
CONCLUSION A total of 19 genera of fungi comprising of 34 species were detected in wheat; some seedborne fungi were saprophytic and others were biotrophic, which could cause seedborne diseases in the field. These fungi infecting seeds could reduce viability of seeds to a different extent.
Acknowledgements This study was funded by National Science and Technology Research in the 10th Five-Year-Plan of China (2004BA525B01).
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Health Testing of Wheat. Danish Government Institute of Seed Pathology for Developing Countries, Hellerup, Denmark. p. 168. McGee D C. 1988. Maize diseases. In: A Reference Source for Seed Technologists. A P S Press, St. Paul, Minnesota, USA. p. 149. McGee D C. 1994. Seed assays for Stewart’s wilt and other seed-borne disease of corn. In: Proceeding of Annual Corn and Sorghum Research Conference 48. American Seed Trade Association, Washington DC. pp. 161-168. Mew T W, Misra J K. 1994. A Manual of Rice Seed Health Testing. International Rice Research Institute (IRRI), Philippines. pp. 13-28. Michail S H, Abou Elseoud M S, Eldin M S N. 1999. Seed health testing of corn for Cephalosporium maydis. Acta Phytopathologica et Entornologica Hungarica, 34,35-41. Neergaard P. 1979. Seed Pathology. The Macmillan Press Ltd., England. Tan Z X, Fang D H, Bai Y F, Zhang H, Lu J P. 2002. Testing of seed-borne fungi in tobacco seeds from Yunnan Province. Journal of Southwest Agricultural University, 24,428-430. (in Chinese) Wang J H, Tylkowska K. 2003. Health evaluation on 22 accessions of common bean germplasm seed.Journal of China Agricultural University, 8,55-60. (in Chinese) Wiese M V. 1987. Compendium of Wheat Diseases. 2nd ed. A P S Press, Minnesota. Wu L M, Zhao L J, Li J Q, Liu X L, Liu Y J. 2004. Testing and identifying of seed-borne fungi in three annual flowers. Seed, 7,3-9. (in Chinese) Wu X H, Liu X L, Liu P F, Wang H M, Li J Q. 2003. Testing of seed-borne fungi of major matermelon varieties in China and disinfectioneffect of several fungicides on seed-borne fungi. Journal of Pesticidology, 5,39-44. (in Chinese) Yang J, Liu X L, Wang H M, Li J Q, Zhang L. 2002. Testing of seed borne fungi of major leguminous plants from China and disinfection effect of several fungicides to seed pathogens. Seed, 1,20-21. (in Chinese) Zhang G Z, Zhang X F. 2002. Fungi detection of American ginseng seeds from Beijing and Northeast area in China. China Journal of Chinese Materia Medica, 27,658-661. (in Chinese)
(Edited by WANG Lu-han)
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Agricultural Sciences in China 2007, 6(6): 682-687
ScienceDirect
June 2007
Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China DUAN Can-Xing, WANG Xiao-ming, ZHU Zhen-dong and WU Xao-fei Institute of Crop Sciences, Chinese Academy of Agricultural Sciencesflhe National Key Facilities f o r Crop Gene Resources and Genetic Improvement (NFCRI), Beijing 100081, P.R.China
Abstract There is a primary understanding of the dominant fungi in wheat seeds conserved in the National Crop Genebank of China (NCGB) and an evaluation of the healthy status of wheat germplasm propagated in different regions. A total of 1465 wheat accessions were detected for seedbone fungi by blotter, agar plate, and wash tests. By blotter test, 17 genera of fungi, including more than 30 species, were detected in 712 wheat accessions from Shaanxi, Hebei, and Qinghai provinces, China. Alternaria was the most frequently detected in wheat seeds from Shaanxi Province, followed by Rhizopus, Penicillium, Aspergillus, Bipolaris, Cladosporium, Gonatobotrys, Chaetomium, and others. Seedborne fungi in wheat seeds from Hebei Province, with relatively high incidence were Alternaria, Rhizopus, Penicillium, Aspergillus, Bipolaris, Cladosporium, and Fusarium. In the seeds from Qinghai Province, Alternaria, Rhizopus, Bipolaris, Cladosporium, and Trichothecium are important seed-borne fungi. The seed germination was reduced substantially when seeds were infected by Fusarium verticillioides (syn. F. moniliforme), Bipolaris nodulosa, and Cladosporium herbarum. Eighteen genera and 25 species of fungi were identified in 353 accessions from Shaanxi Province using the agar plate test. The dominant fungi were Alternaria, Aspergillus, Bipolaris, Gonatobotrys, Penicillium, and Fusarium. The smut fungi, Ustilago tritici, was detected by the wash test in 400 accessions, but it was low in incidence in 300 seed samples from Shaanxi Province (1.3%), and in 100 samples from Hebei Province (2.0%). Totally 19 genera of fungi were detected in wheat seed samples, and some seedborne fungi were saprophytic and others were biotrophic which could cause seedborne diseases in the field.
Key words: wheat germplasm, seedborne fungi, seed health, detection
INTRODUCTION Plant germplasm resources are viewed as the root and foundation for crop production and breeding. They are also indispensably the strategic wealth for survival and development of human. To date, the crop Genebank system has been established in China and 350 000 crop accessions have been conserved in the long-term Genebank and 280000 germplasms in the medium-term
Genebank. The promotion of crop yield and quality greatly depends on the quality of seeds. Seed health has been considered as an attribute of high quality and one of the most important premises for safe conservation. Seedborne fungal, bacterial, and viral pathogens have some deleterious effects on seed, such as, reducing seed viability, vigor and germination capability, shortening longevity of conservation, and causing physiological changes. Furthermore, some seedborne pathogens are also seed-transmitted, which
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence WANG Xiao-ming, Professor, E-mail: [email protected]
02007,CAAS. All @hts resewed. Published by ElsevierLtd.
Testing of Seedborne Fungi in Wheat Germolasrn Conserved in the National Crop Genebank of China
can cause severe diseases in the field after seed movement at a long distance (Neergaard 1979). Wheat is one of the most significantly staple crops in the world. High quality seeds are important to maintain production, improve yield and quality, and conserve germplasm safely. In recent years, the studies on seed health testing have been increasingly deployed in rice, maize, tobacco, fruit, common bean, annual flowers, American ginseng, leguminousplants, and other crops in China (Liu et al. 2000; Li and Hu 2001; Li et al. 2001; Tan et al. 2002; Yang et al. 2002; Zhang G Z and Zhang X F 2002; Wang and Tylkowska 2003; Wu et al. 2003; Wu et al. 2004). However, the health status of wheat seeds conserved in Genebank remains elusive. In this study, three routine techniques of seed health testing are applied to monitor and analyze the fungi that infect wheat accessions conserved in the National Crop Genebank of China. The aim is to raise a theoretic foundation to control seedborne fungi, to improve seed quality, and to prolong the period of conservation.
MATERIALS AND METHODS Experimental materials One thousand four hundred and sixty-five wheat accessions selected randomly from the National Mediumterm Crop Genebank of China, which were propagated in Shaanxi, Hebei, and Qinghai provinces respectively, were provided by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.
Experimental methods Blotter test According to Wang and Tylkowska (2003) and Mew and Misra (1994), three layers of 9.0 cmdiameter white blotter papers soaked in sterile distilled water were placed in petri plates allowing NUV light to penetrate. Twenty-five wheat seeds were placed in each plate and four plates of seeds per germplasm were tested. The seeds were incubated for 7-10 d at 22°C with alternate 12 h periods of NUV light and darkness. The plates were approximately 40 cm below the lights. The seeds were examined under a microscope and the
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fungi were recorded. Affected seeds rate = Number of seeds infected with some fungimotal tested seeds x 100% Affected germplasm rate = Number of samples infected with some fungi/Total tested accessions x 100% Affected range is the percentage that all accessions infected by the same fungus varied to a certain extent. Agar plate test Wheat seeds for testing were pretreated with 1.O% (v/v) sodium hypochlorite for 2 min and then rinsed thrice with sterile distilled water. The sterilized seeds were placed in a 9.0 cm-diameter PDA plate (potato 200 g, glucose 20 g, agar 20 g, distilled water 1 000 mL). A total of three plates (10 seeds per plate) were tested. Seeds were incubated at 25°C for 7- 10 d to allow fungal growth and sporulation with an 8-h NUV light per day (McGee 1994; Mew and Misra 1994; Michail et al. 1999; Li et al. 2001). Wash test Fifty wheat seeds were added to 10 mL sterile distilled water in a 100 mL flask. One drop of sterile Tween 20 was added to the flask and then the flask was vigorously shaken for 10 min. The suspension was transferred to a 10 mL tube and centrifuged at 3 000 r/min for 5 min. The pallets were resuspended in 1 mL Shear’s solution and examined for Tilletia spp. and Ustilago spp. with the help of a microscope (McGee 1988; Mathur and Cunfer 1993).
RESULTS Wheat seedborne fungi tested by blotter test Seven hundred and twelve wheat samples, obtained from Shaanxi, Hebei, and Qinghai provinces were tested and 17 genera of fungi, including more than 30 species were detected. There were 17 genera of fungi comprising of 34 species infecting wheat seeds from Shaanxi Province, 22 species of fungi derived from 14 genera affecting wheat from Hebei Province, and 10 genera of fungi, including 13 species found in wheat seeds from Qinghai Province (Table 1). Results revealed that Altemaria tenuis was the grain-infecting fungi detected most frequently in wheat seeds, followed by Rhizopus stolonifer, Penicillium spp., Alternaria triticina, Altemaria tenuissima, Chaetomium spp., and others with infection percentages of 5 1.00, 21 .OO, 6.00, 2.80,
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1.90, and 1.90,respectively. There were 0.002%seeds affected by Fusarium poae. A. tenuis was most frequently detected among the 7 12 wheat accessions, followed by R. stolonifer, Penicillium spp., A . triticina, Bipolaris nodulosa, A. tenuissima, and Aspergillusflavus with the percentages being 90.20, 45.14, 43.20, 36.00, 26.13,24.11,and 20.80,respectively, whereas, incidence of Stemphylium sp. and E poae were both 0.14%. Affected range by A . tenuis, R . stolonifer. A. jlavus, Monilia tetrasperma, and Penicillium spp. varied to an extent of 1.0-100, 1.0-100, 1.0-89.0,1.0-85.0,and 1.082.0%,respectively. Therefore, it was confirmed that wheat germplasm was susceptible to some fungi, such as Alternaria, Rhizopus, Penicillium, Bipolaris, and Cladosporium. A, f l a w s was carcinogenic and was poisonous for humans and cattle. Germinability of seeds, infected with some seedborne fungi, such as Fusarium verricillioides (syn. F. moniliforme), B. nodulosa, and Cladosporium herbarum, declined significantly and therefore many seeds even lost viability completely. Therefore, it was crucial to control the
fungi for safe conservation of wheat germplasm.
Analysis of health status of wheat accessions from different regions by blotter test Analysis of fungi infecting 712 wheat accessions from different areas, including 28 accessions from Qinghai. 166 from Hebei, and 5 18 from Shaanxi, indicated that the number of fungi species affecting seeds from Shaanxi markedly exceeded those from Qinghai and Hebei (Table 2), which were probably related to the accessions origin and number of samples tested. A. tenuis, R. stolonifer, and Penicillium spp. were frequently found in wheat seeds from Shaanxi, with percentages of 48.9, 23.9, and 7.53 and the affected accessions rates were 87.2, 55.1, and 54.9% in these three species of fungi, respectively. As a whole, wheat accessions from Shaanxi were subject to invasion from eight genera of fungi, comprising of Alternaria, R h i z o p us , Pen i c i 1 1 i M m , A s p e rg i 11u s , B i p o 1 a ris , Cladosporium, Gonatobotlys, and Chaetomium.
Table 1 Incidence of seedborne fungi detected in 712 wheat accessions by the blotter test Fungus
___.__~
Alrernariir rriricinu .4. renuissirnu .4. renuis Asper,qilh niger 4. f l a w s A . cundidu>
Aspergillus sp. Chaeromiurn spp C. Perithecium C. herharum C. macrocarpum C. oxysporium Dictyoarthrinium quudratum Bipolaris hicolor B. cynodonris B. noduloso B. teframrrii Epicoc cum purpurast'ens Fusarium graminearurn F. rnoniliformc F. pour F. remrrccrurn Gonatohorn s africariu Morrilia rerruspernra Nigrosporu cp Penicilliuni spp. Phomo spp Hhizopus srolonifer Stemphylium sp. Trichotlrecrum roseuni Verticilium lateritium
Affected seeds ('h)
Affected accession (%)
2.80 2.00
36.00 26.13 90.20 14.32 20.80 3.87 11.50 9.18 1.29 7.60 3.30 14.80 0.57 12.60 10.52 24.11
51.00 0.57
1.40 0.10 0.68 1.90 0.02 0.17 0.10 0.40 0.01 0.37 0.22 0.70 0.04 0.02 0.02 0.10 0.002 0.04 1.10 0.81 0.03 6.00 0.01 2 I .oo 0.02 0.58 0.03
2.30 1.oo 1.oo 2.58 0.14 2.44 13.63 5.16 I .72 43.20 0.43 45.14 0.14 9.61 I .29
Affected range - in seeds (Yo) 1.00-61.00 1.00-73.00 1.00-100.00 1.00-49.00 1.00-89.00 1.00-4.00 I .oo-22.00 1 .OO-79.00 I .OO-6.00 1.00- 11.00 2.00-18.00 1.00-20.00 1.00-1.00 1 .OO-8.00 1.OO-6.70 I .OO-14.00 1 .oo-3.00 1.OO-4.00 1 .oo-2.00 1.OO-15.00 1.00-1.00 1.00-4.00 1.00-38.00 1.OO-85 .OO I .OO-3 .OO 1.00-82.00 1 .OO-3.00 1.00-100.00 3.00-3.00 1.00-33.00 1.00-6.00
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Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China
The ratios of seeds affected by A . tenuis, R . stolonifer, and A. triticina from Hebei were 67.9, 11.8, and 1.96%, respectively, whereas, the percentages of accessions infected by A . tenuis, A. triticina, and B. nodulosa reached 99.4,44.0, and 33.1%, respectively. There were seven genera of fungi with high incidence, in c ludi n g A 1 t e rna ri a , R h izop u s , Pen ic i 11 i um , Aspergillus, Bipolaris, Cladosporium, and Fusarium. As far as wheat accessions from Qinghai, some seedborne fungi such as Altemaria, Rhizopus, Bipolaris, Cladosporium, and Trichothecium were prevalent. Wheat seeds infected by A. tenuis were most frequent, followed by A . tenuissima and R. stolonifer with the percentages of 79.3, 6.25, and 4.71, respectively, A. tenuis, A. tenuissima, and B. bicolor were detected frequently in accessions with the percentages of 100,95.7, and 78.3, respectively.
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were discovered in 353 wheat accessions from Shaanxi by using the agar plate test (Table 3). Seedborne fungi, which were frequently detected were A . tenuis, R. stolonifer, A . triticina, and A. tenuissima. The percentages of seeds affected by them were 65.3, 33.2, 11.O, and 10.6 and the accessions infected reached up to 99.4, 67.7, 55.8, and 61.2%, respectively. Affected range in accessions by fungi, such as A . tenuis, R. stolonifer, A. triticina, A. tenuissima, and Gonatobotrys africana vaned to a great extent. Six genera of fungi, including Altemaria, Rhizopus, Aspergillus, Bipolaris, Gonatobotrys, and Penicillium were readily detected by the agar plate test. In addition, Pestalotia macrotricha, which was never reported as a seedborne fungus in wheat seeds, was detected with 0.57% of incidence in the accessions tested.
Seedborne fungi examined by wash test Seedborne fungi detected by arga plate test Eighteen genera of fungi, comprising of 25 species,
A hundred wheat accessions from Hebei and 300 from Shaanxi were tested by wash test for Tilletia spp. and
Table 2 Incidence of seedborne fungi detected in wheat accessions from three provinces by the blotter test Shaanxi Province A. triticina A. renuissima A. tenuis A. niger A. f l a w s A. candidus Aspergillus sp. Chaetomium spp C. Perithecium C. herbarum C. macrocarpum C. oxysporium D. quadratum B. bicolor B. cynodontis B. nodulosa B. retramera E. purpurascens F. graminearum F. moniliforme F. poae F. semitectum G. africana M . tetrasperma Nigrospora sp. Penicillium spp. Phoma spp. R. stolonifer Stemphylium sp. T. roseum V. lateritium
3.16 2.13 48.92 0.65 1.80 0.13 0.83 2.44 0.03 0.19 0.12 0.47 0.01 0.30 0.16 0.70 0.04 0.02 0.02 0.12 0.002 0.03 1.42 0.84 0.04 7.53 0.01 23.90 0.01 0.72 0.04
Affected seeds (%) Hebei Qinghai Province Province 1.96 1.07 67.90 0.36 0.02
1.58 6.25 79.32
0.06
0.50
0.07 0.02 0.12 0.01 0.58 0.54 0.94 0.03
0.67 0.75 2.00
0.25 0.04
0.01 0.10 0.01 0.89
0.29
0.35
0.29
11.83
4.71
0.01 0.02
0.50
Affected accession (%) Shaanxi Hebei Qinghai Province Province Province 33.70 28.40 87.22 18.03 27.30 5.16 14.20 12.22 1.72 8.99 4.21 17.01 0.57 10.10 8.22 21.60 2.49 1.14 1.15 3.25 0.19 2.10 17.83 5.93 2.29 54.92 0.57 55.10 0.19 12.20 1.53
44.00 12.70 99.43 4.22 1.81
39.12 95.70 100.00
10.80
30.42
3.01 0.60 6.02 0.60 16.92 18.10 33.10 1.81
34.81 56.50 78.30
17.40 4.35 0.60 3.61 0.60 3.01
26.12
8.43
21.70
13.92
43.50
0.60 0.60
34.90
Affected range in seeds (76) Shaanxi Hebei Qinghai Province Province Province 1.00-61.00 1.OO-73.00 1.00-100 1.00-49.00 1.OO-89.00 1.00-4.00 1.oo-22.00 1.00-79.00 1.00-6.00 1.oo-11.oo 2.00- 18.00 1.00-20.00 1.00 1.00-8.00 1.00-6.70 1.OO-14.00 1.00-3.00 1.00-4.00 1.oo-2.00 1.00-15.00 1.00 1.00-4.00 1.00-38.00 1.00-85.00 1.00-3.00 1.00-82.00 1.00-3.00 1.00-100 3.00 1.OO-33.00 1.OO-6.00
1.00-16.00 1.00-20.00 4.00-100 1.oo-20.00 1.oo
2.00-6.00 3.00- 13.00 70.00-90.00
2.00
1.00-2.00
2.00-4.00 2.00 2.00-4.00 1.00 1.30-10.00 2.00-8.00 1.00-6.00 2.00
1.00-3.00 1.00-3.00 1.00-5.00
1.oo-2.00 1.oo 2.00 2.00-4.00 2.00 9.00-30.00
1 .oo-2.00
2.00-10.00
1.00-2.00
2.00-100
2.00-4.0 1
2.00 2.00
1.00-3.00
-,the fungus was not found.
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Table 3 Incidence of seedborne fungi detected in 353 wheat accessions by agar plate test Fungus
______~-. .___
A. triticina A . tenuissimo A. tenuis A. niger A. Javrrs A. candidus Aspergillu.5 sp. Chaetomium spp. C. lierbar~ni B. noduloso B. tetramerii E. purpurascens F . moniliforme F..semitecrum G. qfricano M rerraspermn N ig rosporo \p . Penicillium \pp. Pesralotia macrorrichu Phoma spp R. rrolonifer Sremphylinm sp. 7 rnseum
Affected seeds (8) I 1 .oo 10.63 65.32 0.59 0.28 0.01 0.1 I 0.05 0.59 I .93 0.05 0.05 0.03 0.18 3.70 0.09 1 .O1 1.88 0.02 0.03 33.20 0.25 0.5 1
Ustilago spp. There were six accessions with Ustilago tritici spores in 400 accessions, and the incidence of Ustilago tritici was 1.30% in 300 accessions from Shaanxi and 2.0070 in 100 accessions from Hebei. Tilletia spp. was not detected in 400 wheat accessions.
DISCUSSION Most samples uere infected by several species of fungi in 1465 wheat accessions tested. The seedborne fungi could damage seeds at a different degree, causing seed shrinkage or color change. Some fungi, such as Alternaria, Aspergillus, Bipolaris, and Fusarium produced different fungal toxins, which could make changes in the chemical ingredients inside the seeds, reduce nutritive value and viability of seeds, and even cause seed death. In addition, some seedborne fungi were also the causal agents of diseases invading the roots, stems, and leaves of wheat. For instance, A . triticina could cause leaf blight and root rot, F. poae and F. verticillioides led to foot rot and scab, Bipolaris spp. caused spot blotch. and Cladosporium rendered blach head mold (Wiese 1987; Lu 2001). The diseases transmitted by seeds resulted in an increase of difficulty in disease control and in the decline of yield and
Affected accession (8) 55.82 61.21 99.40 13.93 7.37 0.28 2.83 1.42 13.02 21.50 1.13 I .42 0.57 5.38 14.70 0.28 9.63 32.63 0.57 0.85 67.72 5.67 6.80
Affected range in seeds (8) 3.33-86.73 3.33-70.00 10.0-96.73 3.33-10.00 3.33-6.73 3.33 3.33-6.73 3.33 3.33-13.33 3.33-33.33 3.33-6.73 3.33 3.33-6.73 3.33 3.33-80.00 30.00 3.33-26.73 3.33-23.33 3.33 3.33 6.70-96.73 3.30-6.73 3.30-23.33
quality. Seed health testing was deployed widely in America, Europe, and some developed countries, and molecular biological techniques were used to detect seedborne pathogens swiftly. In addition to blotter, agar plate, and wash tests, a fluorescent test was also a routine method and its targeted fungus was Septoria nodorum. At present, the blotter test is most widely applied to detect seedborne fungi. Compared with the agar plate, the blotter test was simpler, more economic, and effective in that certain species of fungi were difficult to identify due to hard sporulation. Certainly, there were some advantages of the agar plate test, for example, the tester familiar with cultural characters of the fungal colony could quickly identify the species of the fungus with a naked eye. In China, crops were subject to infection by many pathogens because they were grown in wide areas and different agricultural ecological regions, and thereby some pathogens, of which some can cause hazardous diseases, were transmitted and diffused by infected seeds. The seedborne pathogenic fungi kept long-term survival in seeds, when the seeds were conserved in the Genebank, under conditions of low temperature and relative humidity, which had a harmful effect on germplasm viability and genetic integrity, and also en-
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Testing of Seedborne Fungi in Wheat Germplasm Conserved in the National Crop Genebank of China
gendered a potential threat to agricultural production when these germplasm was planted and propagated after decades. Therefore, seed health testing could provide a scientific foundation for controlling seedborne pathogens in crop propagates, conservation of germplasm in the Genebank, and monitoring the seed’s vigor in a long-term conservation.
CONCLUSION A total of 19 genera of fungi comprising of 34 species were detected in wheat; some seedborne fungi were saprophytic and others were biotrophic, which could cause seedborne diseases in the field. These fungi infecting seeds could reduce viability of seeds to a different extent.
Acknowledgements This study was funded by National Science and Technology Research in the 10th Five-Year-Plan of China (2004BA525B01).
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(Edited by WANG Lu-han)
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