I N T R O D U C T I O N
Leaf blight or fire blight, caused by B o t rytis elliptica (Berk.) Cooke, is one of the most serious diseases of lily, Lilium hybridium Hort., under cool and wet conditions(6, 8, 16). In 1997, approximately 315 ha of lilies started from bulbs were imported from Netherlands and grown for the cut flowers were estimated to have NT$ 836 million of market value in Ta i w a n( 1 ). The central of Taiwan, especially in the Nan-tou and Taichung areas, is a major region for production of lilies. Hsieh and Huang (1998)(16) demonstrated that, under favorable environmental conditions in winter and spring, economic losses due to Botrytis leaf blight disease could reach 100%. Fungicide treatment is a major mean of disease control techniques in lilies. However, after intensive chemical use, dicarboximide and benzimidazole resistant strains of B . e l l i p t i c a have appeared( 4 , 1 5 , 1 9 , 2 4 ). New strategies to control this disease are highly desirable.
Conventional screening for fungicide resistance and e fficacy requires labor intensive methods in the greenhouse. The methods for screening and selecting of fungicides and antagonists could be facilitated. Gould et al. (1996) described
a petal disk assay for screening antagonists to control petunia gray mold caused by B. cinere a( 1 3 ). Hsiang and Chastagner (1991) also used a leaf disk assay to assess virulence of B . c i n e re a and B. tulipae on tulip( 1 5 ). The objective of this study was to establish a leaf-disk assay to screen chemical fungicides and biological antagonists for control of the lily leaf blight.
M ATERIALS AND METHODS
Isolates of Botrytis elliptica
Two single-spore isolates (B061 and B066) of B . e l l i p t i c a were obtained from leaves of diseased oriental lily c v. Casa Blanca at Tien-Chung and cv. Star Gazer at Hsin-She, respectively, in the central areas of Taiwan. Isolates were then maintained on potato dextrose agar (PDA; Difco L a b o r a t o r y, Detroit, MI, USA) and stored at 4℃. To maintain its pathogenicity, the spores were inoculated onto surface-sterilized lily leaves, and then reisolated by harvesting freshly-formed spores from diseased tissues incubated under near UV light (Black Light Blue, F10T8BLB, Sankyo Denki, J a p a n )( 6 ). These two isolates were used in the following assays.
Leaf-disk method for assessment of disease severity of lily leaf blight
caused by Botrytis elliptica
Ting-Fang Hsieh
1and Jenn-Wen Huang
2 , 31. Department of Plant Pathology, Taiwan Agricultural Research Institute, Council of Agriculture, Wu - f e n g , Taichung 413, Taiwan, ROC.
2. Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan, ROC. 3. Corresponding author, E-mail: jwhuang@dragon.nchu.edu.tw; FAX: 04-22851676
Accepted for publication: February 20,2001
A B S T R A C T
Hsieh, T. F., and Huang, J. W. 2001. Leaf-disk method for assessment of disease severity of lily leaf blight caused by B o t rytis elliptica. Plant Pathol. Bull. 10:37-44.
A leaf-disk assay was developed for disease assessment of lily leaf blight caused by B o t rytis elliptica. Oriental hybrid lily (cv. Star Gazer) grown from bulbs in a greenhouse for 1.5 to 2 months was used to prepare leaf disks. The fifth to tenth leaves below the apex were surface-disinfested by dipping entire leaves into 0.1% (v/v) sodium hypochlorite three times and rinsed in sterile water after each dip. Leaf disks (15-mm diameter) were then perforated from surface-disinfested leaves with a cork borer, and fully submerged in fungicide or antagonist solutions. The disks (8 per replication) were placed abaxial surface side up in petri dishes containing a piece of 60-mm-diameter sterilized filter paper. Two milliliters of sterile water were added to the filter paper to maintain moisture. Spore suspension of B. elliptica was adjusted to 105spores per ml and was sprayed over the disks as inoculum, then the disks were incubated in 20℃ incubator without light. After 3 days incubation, disease severity was assessed as percent necrotic area. The results of the leaf-disk assay were consistent and agree with that of whole plant assays in the greenhouse.
Inoculum pre p a r a t i o n
To prepare fungal inoculum, the two isolates of B . e l l i p t i c a were grown on autoclaved lily leaves or petals for 7 to 10 days at 20 ℃ under near UV light. Spores were
harvested from fungal colonies with sterile forceps, which were then swirled in 5 ml sterile water to release the spores. The spore suspensions were centrifuged at 3000x g for 10 min, supernatant was discarded, and 5 ml sterile water was added to the tube. This collection step was repeated three times. Spores were then counted with a hemacytometer, and spore suspension was adjusted to a desired concentration( 1 6 ).
Isolation and preparation of bacterial antagonists
Lily leaves used for the isolation of bacterial antagonists were obtained from several commercial nurseries in central areas of Taiwan. Four to five grams of whole leaves were cut into 0.5 x 1 cm pieces, rinsed in distilled water and shaken at 100 rpm by shaker for 1 h at room temperature (approximately 25℃). Ten-fold dilutions of the leaf-washes were prepared immediately, and 100μl of the dilutions were plated onto nutrient agar (NA; Difco Laboratory, Detroit, MI, USA) with four replicates. After 4 days, single colonies were isolated and streaked on NA to obtain pure cultures. After preliminary trials with dual cultures of bacteria confronting B . e l l i p t i c a mycelium, the unidentified bacterial isolates 24-2-2, 4922, BNA12, BPDAS21, DNYDA602, and Y N Y D A -21 were selected for leaf-disk tests. The bacterial isolates were cultured in nutrient broth (NB; Difco Laboratory, Detroit, MI, USA) at room temperature for 4 days on shaker with shaking speed at 100 rpm. Bacterial concentrations were adjusted by adding sterile nutrient broth to attain an optical density of 0.38 at 600 nm with spectrophotometer (UV- 1 2 0 1 , Shimadzu Co., Kyoto, Japan), which approximates a concentration of 108cfu of bacteria ml- 1(as determined by a standard curve).
Fungicide pre p a r a t i o n
The following seven fungicides recommended for control of gray mold in Taiwan were chosen to determine the control efficacy of lily leaf blight. Those were procymidone, 50% wettable powder (WP) [ N-(3',5'-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide, Sumitomo Chemical Taiwan, Taipei ], vinclozolin, 50% WP [ 3-(3,5-d i c h l o r o p h e n y l ) - 5 - e t y e n y l - 5 - m e t h y l - 2 , 4 - o x a z o l i 3-(3,5-d i n e 3-(3,5-d i o n e , B A S F, Ludwigshafen, F. R. Germany ], thiophanate-methyl, 70% WP [ dimethyl-4,4'-O-phenylene-bis (3-thioallophanate), B A S F, Ludwigshafen, Germany ], iprodione, 23.7% WP [ 3( 3 , 5 d i c h l o r o p h e n y l ) N i s o p r o p y l 2 , 4 d i o x o i m i d a z o l i d i n e 1 -carboxamide, Rhone-Poulenc, Paris, France ], dichlofluanid, 50% WP [ N-dichlorofluoromethylthio-N', N'-dimethyl-N'-phenylsulfamide, Euparen, Bayer AG, Leverkusen-Bayerwerk, Germany ], mancozeb, 80% WP [ Mn+ + 1 6 % , Z n+ + 2%, ethylene-bis (dithiocarbamate) polymeric complex
62%, Du Pont, Wilmington, DE 19898, USA ], and carbendazim, 50% WP [ methyl-2-benzimidazole carbamate, B A S F, Ludwigshafen, F. R. Germany ]. All fungicides were diluted to field recommended rates. Procymidone, vinclozolin and dichlofluanid were diluted to 1500 fold. Thiophanate-methyl, iprodione and carbendazim were diluted to 1000 fold and mancozeb was diluted to 500 fold.
E ffects of spore concentration, temperature, inoculum
d roplet size and leaf position on the disease severity
of Botrytis leaf blight using a leaf disk assay
Oriental hybrid lily (Lilium oriental) cv. Star Gazer was grown from bulbs in the greenhouse for 1.5 to 2 months and used to prepare leaf disks. The fifth to tenth leaves below the apex were surface-disinfested by dipping entire leaves in 0.1% sodium hypochlorite three times for 3 min, rinsed in three changes of sterile water and blotted dry on sterilized paper towels. Leaf disks (15-mm diameter) were cut from surface-disinfested leaves with a cork borer. The disks (eight pieces per dish) were placed adaxial surface down in petri dishes (90 mm in diameter) containing a 60-mm-diameter piece of sterilized filter paper. Two milliliters of sterile water were added to the filter paper to maintain moisture. A series of experiments were conducted to determine the influence of spore concentration, temperature, inoculum droplet size and leaf position on disease development. A spore suspension prepared as described above and adjusted to 10, 102, 103, 104 and 105 spores ml- 1 was applied (Sigma spray unit, Sigma Chemical Co, St. Louis, MO, USA) to leaf disks (about 25μl
per disk). Leaf disks were incubated at 20℃ in darkness for 3, 4 and 5 days and percent infected area per leaf disk was recorded. The effect of temperature was evaluated by incubating leaf disks which had been inoculated with 25μl of
1 05spores ml- 1per leaf at 8 to 32℃, interval 4℃, in darkness for 3 days, and each was examined for the percent infected area. To determine the effect of the droplet size of the inoculum on the disease development, spore suspension (105 spores ml- 1) was applied to leaf disks with the Sigma spray unit (Sigma Chemical Co., St. Louis, MO, USA) to form droplet sizes of approximately 0.1, 0.2, 0.25, 0.3 and 0.4 mm in diameter. The leaf disks were then incubated at 20℃ i n
darkness, and infected area per leaf disk was assessed 3 days l a t e r. Since leaf position was found to influence disease development in a preliminary test, all leaves of each plant were stripped and leaf disks were prepared. After inoculation and incubation as described above, disks were evaluated by counting percent-infected area. All experiments were repeated at least twice.
Leaf disk assays for screening antagonists and fungicides
Leaf disks were dipped thoroughly either in seven fungicides or six solutions of bacterial antagonists prepared as described above for 10 min. After the surfaces were air dried,the leaf disks were placed with adaxial surface down in petri dishes containing a piece of 60-mm-diameter sterilized filter p a p e r. Two milliliters of sterile water were added onto the filter paper to maintain moisture. A spore suspension adjusted to 105 spores ml- 1was sprayed over the disks to form inoculum droplets of up to 0.3 mm in diameter. The leaf disks were then incubated at 20℃ in darkness, and the percentage of infected area was evaluated after 3 days.
Whole plant assays
To compare the relationship of the response of leaf disks assays and that of whole plant assays, the fungicides and antagonists (prepared as above) were tested for control of Botrytis leaf blight on whole lily plants (cv. Star Gazer). Single lily bulbs were grown in 18-cm-diameter plastic pots with non-sterilized nursery soil. The plants from lily bulbs were kept in a greenhouse at 24±4℃. After growth of 1.5 to 2 months, entire lily plants were sprayed with fungicides and antagonists prepared as described above. After air- d r i e d , plants were sprayed by spore suspension of B. elliptica w i t h concentration of 105spores ml- 1. Each treatment had four replicates and both tap water and nutrient broth sprays served as controls. The inoculated plants were sealed in plastic bags to maintain humidity for 2 days and then were moved into growth chambers of 20℃ with 16 h of light period at light
intensity of 3500 lux (Digital illumination meter, DX-200, Jing Chen Instruments Co., Taichung 404, Taiwan). Disease severity was analyzed 7 days after inoculation. The lesion numbers of the fifth to eighth leaves were counted. The experiment was repeated twice.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA ) , and the means were separated by Duncan's multiple range t e s t .
R E S U LT S
E ffects of spore concentration, temperature, inoculated
d roplet size and leaf position on the disease severity
of Botrytis leaf blight with leaf disk assays
In spore concentration tests, a rating of 80% lesion area on leaf disks was observed 3 days after inoculation with 105 spores ml- 1. This result was equivalent to 5 days after inoculation with 104spores ml- 1(Fig. 1). However, the data of lesion area were less variable when leaf disks were inoculated with 105spores ml- 1spore concentration. Most serious disease development was observed in inoculated leaf disks incubated under lower temperatures (12-24℃). No lesions were observed at 32℃. The optimum temperature for disease development was 20℃ (Fig. 2). Droplets of inoculum up to 0.4 mm in diameter were positively related to lesion area expansion. The infected area on leaf disks up to 80% was
obtained at the drop size of inoculum up to 0.3 mm in diameter after 3 days inoculation (Fig. 3). When the diameter of inoculum droplets were up to 0.4 mm or over, the droplets would easily fuse together. In this condition, the inoculum was difficult to distribute uniformly on the leaf disks. Leaf disks from leaves of any position on plant could be infected by B. elliptica. The fifth to tenth leaf below the apex were less variable in disease development (Fig. 4). The percentage of lesion area on leaf disks was positively related (r=0.717, p<0.01) to lesion number per leaf on whole plants in greenhouse tests (Fig. 5).
A f t e r e v a l u a t i o n o f a l l f a c t o r s a f f e c t i n g d i s e a s e development, a standardized leaf-disk assay was devised as follows (Fig. 6). Lilies grew from bulbs in a greenhouse for 1.5 to 2 months were used to prepare leaf disks. The fifth to tenth leaves below the apex were surface-disinfested by dipping entire leaves in 0.1% sodium hypochlorite three times and rinsing it in three changes of sterile water. Leaf disks (15-mm diameter) were cut from surface-disinfested leaves by means of a cork borer for advanced treatment. The disks (8 per replication) were placed abaxial up in petri dishes (90 mm in diameter) containing a piece of 60 mm in diameter of sterilized filter paper. Two milliliters sterile water was added to the filter paper to keep moisture. A spore suspension adjusted to 1 x 105spores ml- 1was sprayed over the disks to form inoculum droplets of up to 0.3 mm in diameter. They were then incubated at 20℃ in darkness, with disease severity assessed after 3 days. The percentage of necrotic area on each leaf disk was recorded.
Fig. 1. The relationships between the inoculated concentrations
of B o t rytis elliptica and disease development of lily leaf blight on leaf disks at 20℃ for 3 and 5 days.
Leaf disk assays for screening antagonists and fungicides
In screening antagonists, the percentage of infected area per leaf disk was lower after leaf disks were treated with 108 cfu ml- 1 suspension of bacteria such as B-NA-12, B-PDAS-21, D-NYDA-602 or Y-NYDA-B-PDAS-21, and tested against twoisolates of B. elliptica ( Table 1). In fungicides trials compared to water-treated leaf disks inoculated with B061 or B066 isolates of B. elliptica, all fungicides, especially iprodione, dichlofluanid and mancozeb, significantly reduced the percentage of necrotic area on leaf disks (Table 2). Among
Fig. 3. The relationships between droplet size of inoculum
( 1 05spores ml- 1) and infected area by B o t rytis elliptica on lily leaf disks at 20℃ after 3 days incubation. The trials were carried out twice (Test I and II).
Fig. 4. The influence of leaf position of lily ( L. oriental
hybrid cv. Star Gazer) on disease development of lily leaf blight caused by B o t rytis elliptica at 20℃ for 3 days.
Fig. 5. The relationship between infected area on leaf disks (3
days) and lesion number per leaf on whole plant assays (7 days) after inoculation with B o t rytis elliptica at 20℃.
Fig. 2. The effect of temperatures on disease development of lily
leaf blight caused by B o t rytis elliptica for 3 days after 105 s p o r e s m l- 1inoculation. The trials were carried out twice (Test I and II).
Fig. 6. Leaf disk method. (A) The fifth to tenth leaves below the apex of Oriental hybrid lily (cv. StarGazer) grown from bulbs in
a greenhouse for 1.5 to 2 months were used to prepare leaf disks. Leaf disks (15-mm diameter) were cut from surface-d i s i n f e s t e d leaves with a cork borer. (B) The disks (8 per dish) were placed abaxial surface up in petri dishes containing a piece of 60-mm-diameter sterilized filter paper. Two milliliters of sterile water were added to the filter paper to maintain moisture. (C) Spore suspension was sprayed over the disks to form inoculum droplets of up to 0.3 mm in diameter. (D) The inoculated disks were incubated at 20℃ in darkness. (E) Water soaked lesions appeared on the surface of leaf disks 16 h after inoculation. (F) Disease
tested fungicides, thiophanate-methyl (dicarboximide fungicide) and carbendazim (benzimidazole fungicide) showed lowest efficacy in decreasing the percentage of lesion area on leaf disks inoculated with B061 and B066 isolates (Table 2).
Whole plant assays
Antagonists such as B-NA-12, B-PDAS-21, D-NYDA-602 and Y-NYDA-21 effectively reduced the lesion number of lily leaf blight on leaves (Table 1), but isolates 24-2-2 and 49-2-2 were not effective in decreasing lesion number. In fungicide trials, dichlofluanid 50% WP and mancozeb 80% WP were the most effective for controlling lily leaf blight ( Table 2). The effect to reduce the lesion number on whole plants by dicarboximide (procymidone, vinclozolin and iprodione) and benzimidazole (carbendazim and thiophanate-methyl) fungicides was less effective than that of dithiocarbamate (mancozeb) and sulphamide (dichlofluanid) fungicides (Table 2). The response of whole plants assay was in agreement with the results obtained from the leaf disk assays, except for two treatments (bacterium 49-2-4 and fungicide vinclozolin 50% WP) against isolate B066.
D I S C U S S I O N
Lily leaf blight, caused by B o t rytis elliptica, is a severe disease on ornamental lily in Taiwan as well as many other countries during low temperature season( 6 , 1 6 ). Doss and his colleagues produced a series of reports on spore production of B. elliptica( 6 ), infection process of the pathogen on Easter lily (8) and screening varieties resistance of lily to the pathogen with a detached leaf method( 7 ). In an earlier study, we found that preparation of materials and lily plants were laborious and expensive( 1 6 ). For this reason, we hope to develop a bioassay afforded to save materials and allow screening of many treatments at one time be possible. Thus, leaf disks have been used instead of whole plants in many previous experiments such as detection of disease resistance ( 2 , 3 , 5 , 11 , 1 4 , 1 8 , 2 1 ), detection of herbicide tolerance ( 1 2 ), determination of host-pathogen interactions( 2 2 ), genetic transformation systems( 2 0 ), entomological researches(17, 23) and botanical researches( 2 6 , 2 7 ). In this study, we demonstrated that the leaf disk assay might accurately predict the response of lily to B. elliptica under control conditions. Result showed that using leaf disks to screen antagonists and fungicides for Table 2. Comparison of leaf disks and whole plant assays on evaluating the efficiency of fungicides on control of lily leaf blight caused by B o t rytis elliptica.
F u n g i c i d e D i l u t e d
Leaf-disk assay 1 Whole plant test 2
f o l d Lesion area (%) Lesion number per leaf
B 0 6 13 B 0 6 6 B 0 6 1 B 0 6 6 Carbendazim, 50% WP 1 0 0 0 59.3 b4 56.0 b 115 b 80 bc Dichlofluanid, 50% WP 1 5 0 0 1.8 d 0.0 c 9 d 9 d Iprodione, 23.7% WP 1 0 0 0 10.4 d 2.8 c 60 c 55 c Mancozeb, 80% WP 5 0 0 6.5 d 4.6 c 4 d 8 d Procymidone, 50% WP 1 5 0 0 32.6 c 13.5 c 77 c 61 bc Thiophanate-methyl, 70% WP 1 0 0 0 55.2 b 38.1 b 102 b 87 bc Vinclozolin, 50% WP 1 5 0 0 27.5 c 9.4 c 62 c 95 ab Wa t e r 82.4 a 78.7 a 152 a 124 a
Footnotes are the same as Table 1.
Table 1. Disease severity of Botrytis elliptica on leaf disks and whole plants of lily after challenged with different microorganisms
M i c r o o rg a n i s m Leaf disk assay
1 Whole plant test 2
(Isolate designation) Lesion area (%) Lesion numbers per leaf
B 0 6 13 B 0 6 6 B 0 6 1 B 0 6 6 2 4 - 2 - 2 79.38 a4 83.75 a 94.5 a 85.0 abc 4 9 - 2 - 4 78.75 a 70.00 b 83.0 a 105.4 a B - N A - 1 2 48.75 b 32.50 cd 53.7 b 51.3 bc B - P D A S - 2 1 55.63 b 29.38 cd 31.4 c 48.8 c D - N Y D A - 6 0 2 56.88 b 40.63 c 40.6 bc 44.8 c Y- N Y D A - 2 1 60.00 b 25.63 d 38.9 bc 45.7 c CK1(Nutrient broth) 85.63 a 85.00 a 102.5 a 89.4 ab CK2 (Wa t e r ) 80.50 a 85.00 a 97.7 a 116.2 a 1 .
A spore suspension adjusted to 105 spores ml- 1was sprayed over the disks to make the droplet size of inoculation about 0.3 mm in
d i a m e t e r, then petri dishes were incubated at 20℃ in darkness and the percentage of infected area on leaf disks was recorded at the 3rd day.
2 .
The average numbers of disease lesion on fifth to eighth leaves of the whole plant were counted 7 days after inoculation in the greenhouse.
3 .
Isolates B061 and B066 of B. elliptica were obtained from diseased lily plants.
4 .
control of lily leaf blight was highly correlated with results from whole plant assays.
We used a leaf disk assay to evaluate the efficacy of fungicides rapidly. Our results showed that the fungicides recommended for grey mould control such as procymidone, vinclozolin, iprodione (dicarboximide fungicides), thiophanate-methyl and carbendazim (benzimidazole fungicides) had lower efficacy in controlling lily leaf blight ( Table 2). In fungicides tested, only dichlofluanid and mancozeb showed high efficacy in controlling lily leaf blight. H o w e v e r, more isolates of B. elliptica would have to be screened for their sensitivity. The leaf disk assay presented here could provide a quick and simplified method to pre-screen the fungicides for B. elliptica c o n t r o l .
Biological control is one of several approaches to decrease the usage of chemicals. Bacterial antagonists have been used to suppress the disease progress of grey mould on many crops( 1 0 , 1 3 , 2 5 ). Elad (1990) demonstrated that potential antagonists could be isolated from native habitats of pathogens and could survive, grow and show antagonism to pathogens in that habitat( 9 ). Therefore, The candidate isolates of potential antagonists in this study were obtained from leaves of field-grown lily plants. Some of them showed the potential in controlling lily leaf blight in the leaf disk assays and whole plant trials. Using leaf-disk assays could minimize the number of samples needed, and still reflect the response of whole plant assays. Leaf-disk assays not only saved plant material and trial space, but also shortened the time required to screen fungicides and potential antagonists.
A C K N O W L E D G E M E N T S
The authors would like to thank Dr. T. Hsiang, University of Guelph, Canada for his advice and suggestions on the manuscript and Mr. C. C. Chang for his technical assistance. This study was conducted as part of a research project supported by the grants NSC88-2317-B-005-001 and NSC89-2317-B-055-005 from National Science Council of ROC.
L I T E R ATURE CITED
1 . Anonymous. 1998. Taiwan Agricultural Yearbook, 1998 ed. Department of agriculture and forestry, Ta i w a n Provincial Government. Nan-tou, Taiwan. 398pp.
2 . Arabi, M. I., Sarrafi, A., Barrault, G., and Albertini, L. 1991. An improved technique for determining net blotch resistance in barley. Plant Dis. 75: 703-706.
3 . B u s s e y, M. J., and Stevenson, W. R. 1991. A leaf disk assay for detecting resistance to early blight caused by Alternaria solani in juvenile potato plants. Plant Dis. 75: 3 8 5 - 3 9 0 .
4 . C h a s t a g n e r, G. A., and Riley, K. 1990. Occurrence and control of benzimidazole and dicarboximide resistant
B o t rytis spp. on bulb crops in western Washington and Oregon. Acta Hortic. 266: 437-445.
5 . Cohen, R. 1993. A leaf disk assay for detection of resistance of melons to S p h a e rotheca fuliginea race 1. Plant Dis. 77: 513-517.
6 . Doss, R. P., Chastagner, G. A., and Riley, K. I. 1984. Techniques for inoculum production and inoculation of lily leaves with B o t rytis elliptica. Plant Dis. 68: 854-856. 7 . Doss, R. P., Chastagner, G. A., and Riley, K. I. 1986.
Screening ornamental lilies for resistance to B o t ry t i s e l l i p t i c a. Scientia Hortic. 30: 237-246.
8 . Doss, R. P., Christian, J. K., and Chastagner, G. A. 1988. Infection of Easter lily leaves from conidia of B o t ry t i s e l l i p t i c a. Can. J. Bot. 66: 1204-1208.
9 . Elad, Y. 1990. Reasons for the delay in development of biological control of foliar pathogens. Phytoparasitica 18: 9 9 - 1 0 5 .
1 0 . Elad, Y., Kohl, J., and Fokkema, N. J. 1994. Control of infection and sporulation of B o t rytis cinerea in bean and tomato by bacteria and fungi. Eur. J. Plant Pathol. 100: 3 1 5 - 3 3 6 .
11 . Eskes, A. B. 1982. The use of leaf disk inoculations in assessing resistance to coffee leaf rust ( H e m i l e i a v a s t a t r i x). Neth. J. Plant Pathol. 88: 127-141.
1 2 . Gawronski, S. W., Callihan, R. H., and Pavek, J. J. 1977. Sinking leaf-disk test for potato variety herbicide tolerance. Weed Sci. 25: 122-127.
1 3 . Gould, A. B., Kobayashi, D. Y., and Bergen, M. S. 1996. Identification of bacteria for biological control of B o t ry t i s c i n e rea on petunia using a petal disk assay. Plant Dis. 80: 1 0 2 9 - 1 0 3 3 .
1 4 . Hamelin, R. C., Ferriss, R. S., Shain, L., and Thielges, B. A. 1994. Prediction of poplar leaf rust epidemics from a leaf-disk assay. Can. J. For. Res. 24: 2085-2088.
1 5 . Hsiang, T., and Chastagner, G. A. 1991. Growth and virulence of fungicide-resistant isolates of three species of B o t ry t i s. Can. J. Plant Pathol. 13: 226-231.
1 6 . Hsieh, T. F., and Huang, J. W. 1998. Factors aff e c t i n g disease development of Botrytis leaf blight of lily caused by B o t rytis elliptica. Plant Prot. Bull. 40: 227-240. 1 7 . Knight, A. L., Beers, E. H., Hoyt, S. C., and Riedl, H.
1990. Acaricide bioassays with spider mites (Acari: Tetranychidae) on pome fruits: Evaluation of methods and selection of discriminating concentrations for resistance monitoring. J. Econ. Entomol. 83:1752-1760.
1 8 . Lefevre, F., Pichot, C., and Pinon, J. 1994. Intra- and interspecific inheritance of some components of the resistance to leaf rust (Melampsora larici-populina K l e b . ) in poplars. Theor. Appl. Genet. 88: 501-507.
1 9 . Migheli, Q., Aloi, C., and Gullino, M. L. 1990. Resistance of B o t rytis elliptica to fungicides. Acta Hortic. 266: 429-4 3 6 .
2 0 . Nehra, N. S., Chibbar, R. N., Kartha, K. K., Datla, R. S. S., Crosby, W. L., and Stushnoff, C. 1990. Genetic
transformation of strawberry by A g ro b a c t e r i u m tumefaciens using a leaf disk regeneration system. Plant Cell Rep. 9: 293-298.
2 1 . Rao, M. N., Siegel, M. R., Ferriss, R. S., Nesmith, W. C., Wiglesworth, M. D., Burton, H. R., Reuveni, M., Tu z u n , S., and Kuc, J. 1989. Relationships between susceptibility of field-grown burley tobacco to blue mold and contents of duvatrienediols. Phytopathology 79: 267-270.
2 2 . Sackston, W. E., and Vimard, B. 1988. Leaf disk immersion (LDI) inoculation of sunflower with Plasmopara halstedii for in vitro determination of host-pathogen relationships. Plant Dis. 72: 227-229.
2 3 . Shipp, J. L., and Whitfield, G. H. 1991. Functional response of the predatory mite, Amblyseius cucumeris (Acari: Phytoseiidae), on western flower thrips,
Frankliniella occidentalis (Thysanoptera: Thripidae). Environ. Entomol. 20: 694-699.
2 4 . Skrzypczak, C. 1992. Evaluation of fungicides and Atpol e ffect on control of B o t rytis elliptica (Berk.) Cooke on field grown lilies. Acta Hortic. 325: 793-796.
2 5 . Sobiczewski, P. and Bryk, H. 1996. Biocontrol of B o t ry t i s c i n e rea and Penicillium expansum on postharvest apples by antagonistic bacteria. Acta Hortic. 422: 344-345. 2 6 . S p o m e r, L. A. 1972. Evaluation of edge injection errors in
the floating leaf disk method of measuring leaf tissue water deficit. Plant Physiol. 49: 1027-1028.
2 7 . Wi s s e m e i e r, A. H., and Horst, W. J. 1991. Simplified methods for screening cowpea cultivars for manganese leaf-tissue tolerance. Crop Sci. 31: 435-439.
摘 要
謝廷芳1、黃振文2 , 3. 2 0 0 1. 利用葉片圓盤法評估百合灰黴病的罹病度 植病會刊1 0 : 3 7 - 4 4. (1 .台中縣霧峰
鄉 行政院農委會農業試驗所； 2 . 台中市 國立中興大學植物病理系； 3 . 聯絡作者，電子郵件 :
jwhuang@dragon.nchu.edu.tw ; 傳真 : 04-22851676)
採取露蕾期之葵百合 ( c v. Star Gazer) 葉片，以口徑15 mm之打孔器切取葉片圓盤，評估灰黴病菌
[ B o t rytis elliptica (Berk.) Cooke ] 為害百合葉片的情形，包括決定接種源的濃度、溫度對病害發生之
影響、噴霧接種之水珠大小、植株葉位之感病性，及葉片圓盤分析法與整株百合罹病之相關性。建 立一個葉片圓盤分析的標準方法：即摘取成熟百合植株之第五至十位葉，以 0 . 1 %次氯酸鈉表面消毒 後，經無菌水漂洗三次，晾乾後以口徑 15 mm打孔器切取葉片圓盤，置於平舖一張 6公分之濾紙且含 2毫升無菌水之9公分培養皿中，每皿置放 8片葉片圓盤，然後接種每毫升含 1×1 05個灰黴病菌之胞子 懸浮液，使圓盤上覆一層大小約 0.3 mm之水珠，覆以皿蓋，移置於 2 0℃之定溫箱中觀察病勢的進 展。在室內利用葉片圓盤分析法篩選百合灰黴病菌的防治藥劑與拮抗微生物，結果與溫室測試整株 植物之結果相當一致。葉片圓盤分析法不但可減少植材與葉片用量外，並可節省試驗空間與時間， 且可預先篩選出防治百合灰黴病菌的藥劑與拮抗微生物，以供溫室或田間進一步試驗之用。 關鍵詞：百合、灰黴病、葉片圓盤法、拮抗微生物、藥劑
