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1 Department of Horticulture National Chung Hsing University Master Thesis (Yau-Shiang Yang) (Fuh-Jyh Jan) Studies on Growth of Healthy Seedlings and Virus Detection Techniques in Pear (Ai-Jeng Kuo) June, 2002

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4 Pyrus pyrifolia Nakai cv. Hengshan ( 1991)

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6 (Pyrus communis L.) (P. bretschneideri Rehd.) (P. pyrifolia Nakai) (P. ussuriensis Maxim.) (P. sinkiangensis Yu) (P. phaeocarpa Rehd.) (P. pseudopashia Yu) (P. betulaefolia Bge.) (P. calleryana Decne.) (P. kawadamii Hayata) (P. lindleyi Rehd.) ( 1996) 1,600 ( 1994) 73

7 ( 1979 Lin et al., 1987 ; 1990) 1960 ( )

8 ( 1992) Hayward Bruno Monastra and Chiariotti, 1994 Albarino

9 Grenan, Martinez, 1995 Matthews, 1991 immune infectible nonhost host resistant susceptible (genome) (moving protein) (plasmodesmata) Tobacco mosaic virus (TMV)

10 RNA Geminivirus Geminivirus single strain DNA virus bipartie genome Carrington et al., 1996 (Balachandran et al., 1997) Reinero and Beachy, 1989 Krause and Weis, 1991, 1993 Eupaorium makinoi TMV 64 Krause and Weis, 1991 Reinero Beachy (1989) TMV Cucurbita pepo L. Cucumber mosaic virus (CMV) ADP-glucose pyrophosphorylase total starch hydrolase (Tecsi et al., 1994) Shalitin Wolf (2000) Cucumis melon L. CMV stachyose CMV

11 Tomato masic virus (TmMV) 17% (Maklad et al., 1996) (Khurana, 1979 Kar et al., 1979) Aramburu (1995) Cymbidium mosaic virus (CyMV) Chia and Chan, 1992 Chia and He, 1999 Beta vulgaris L. Beet yellows virus (BYV) 71 Clover et al., (Edwards et al., 2001) 7 Apple chlorotic leaf spot virus (ACLSV) Apple stem pitting virus (ASPV) Apple stem grooving virus (ASGV) Apple mosaic virus (AMV) Cherry rasp leaf virus (CLRV) Tulare apple mosaic virus (TAMV) Tomato ringspot virus (TmRSV) 7 ACLSV ASPV ASGV

12 (Jones and Aldwinkle, 1990) 1 ACLSV ( 2001) ASPV ( 2001) ASGV Gella (1989) Agua de Aranjuez ASPV 79 Engel (1990) Lucas 73-81% 36-46% Wang and Hu, 1980 Havel and Novak, 1985 Fukui et al., 1989

13 (indicator plants) (Chenopodium album L.)

14 1994 (inclusion) 30 (Goodman et al., 1986) Immuneosorbent Electron Microscopy, ISEM (Miller, 1992)

15 (Serological assays) (Sherwood, 1994 Nidolaeva et al., 1995) (SDS-immunodiffusion test) (Enzyme-Linked Immunosorbent Assay, ELISA) (Immuno-printing test) 0.5 cm (Persoon, 1992) DBIA (Dot Blot Immunoassay) DTBIA (Direct Tissue Blot Immunoassay) DBIA ELISA

16 (Chia and Chan, 1992) DTBIA DTBIA Polymerase Chain Reaction, PCR 20 (primer) Taq DNA polymerase (Tsuneyoshi and Sumi, 1996) RNA 3' primer DNA PCR (Kinard et al., 1996 Rush et al., 1994) (Nucleic acid hybridization) complimentary hybridization (Sherwood, )

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18 cm cm 2. (1). (2).

19 (3) Inskeep (1985) 3 10 ml 4 24 (HITACHI U-2000 spectrophotometer) 647 nm nm (17.9A A ) V 1/4S V S (4). (5). RT-PCR ACLSV ASPV ASGV m 1 m cm

20 2. (1). A B. C. D. (2) A. B. Delta-T Dias 1800 C D. 0.2 cm 0.5 cm

21 1.

22 2. RNA degenerate primers Genbank ACLSV ASPV ASGV 2 2. Genbank (degenerate primers) Table 2. Search public virus sequences from Genbank database, and select high conserve block, design degenerate primers. Virus Degenerate primers ASGV FJJ (5 primer): 5 -GTCATCTGATAAGACC FJJ (3 primer): 5 -GTTAGGAGTCGTGTGA ACLSV FJJ (5 primer):5 -AACTCTGGAGAACCACC FJJ (3 primer):5 -TAY(C/T)ATGCCTGATGGT(A/G)GT ASPV FJJ (5 primer): 5 -AGGGTGTAGTTGCTTA FJJ (3 primer): 5 -TGATACGGTCGTACTT 1. RNA -80 RNA ( Napoli, et al., 1990) mg 1.5ml 500 l ( 100 mm NaCl 10 Mm Tris-Cl,pH7.5 1mM EDTA 1% SDS)

23 l phenol vortex 250 l chloroform, vortex 3. (14000rpm) 5 5M LiCl min, pellet 100 l,0.3m NaOAc pellet l 100% , 70% pellet ml RNA 30 µl DEPC-dH 2 O 30 water bath RT-PCR RT-PCR RNA 2 µl (OD 260nm ) 3' 2 µl ( 200 ng/µl) DEPC-dH 2 O 13 µl spin down water bath mm dntp 1 µl 100 mm DTT 3 µl (Epicentrel) 10X MMLV RT buffer 2.5µl (Epicentrel) MMLV-RTase 1µl ( 200 U/µl) 40

24 U/µl RNasin (10,000U MADISON WI USA) 0.5 µl RT-PCR PCR RT-PCR DNA 4.5 µl 3' 1 µl 5' 1 µl dh 2 O 82 µl 1 mm dntp 1 µl 10X Taq DNA buffer 10 µl 1U/1µl Polymerase 0.5 µl PCR PCR 10 µl 0.8 agarose ethidium bromide UV box (Model TC-321A) ACLSV ASPV ASGV RT-PCR

25 3

26 ( ) cm cm g 70.6 g g cm

27 cm µg/cm µg/cm µg/cm µg/cm µg/cm µg/cm RT-PCR

28 ( ) 1. (1) cm cm cm g g g 33 (2) cm cm 11.2

29 (3) µg/cm µg/cm µg/cm µg/cm µg/cm µg/cm 2 (4) ACLSV ASPV (1) g 303 g g cm cm g 29.6 g

30 6.7 g 4.1 g cm cm g 18.7 g g (2) g g g g (3) m 30 cm 75.4 g 54.1 g 25 g 14 g 30

31 (4) RT-PCR 0.8 agarose gel 12 FJJ , bp FJJ , bp FJJ , bp FJJ , bp TOPO Cloning NCBI ACLSV ASPV ASGV

32 ASGV 10 ACLSV 5 ASPV ACLSV ASGV 7 ASPV ASGV 2 ACLSV ASPV ASGV ASGV 7 ACLSV 3 ASPV ACLSV ASGV 7 ASPV ASGV ASGV 4 ACLSV 1 ASPV ACLSV ASGV

33 ASGV ASGV 10 ACLSV 1 ASPV ACLSV ASGV 10 1

34 (1999)

35 3 Robinson RT-PCR ACLSV ASPV shoot tip (apical meristem) (subapical meristem) rib meristem Wang and Hu, auxin

36 ACLSV ASPV ASGV phenolic compound nucleic acid phenolic complex phenol chloroform phenol chloroform ACLSV ASPV ASGV 1-2

37 titer RT-PCR RT-PCR ASGV ASGV ACLSV 30 ASPV

38 (Kinard, 1996 Kundu, 2000 Krizbai et al., 2000) RT-PCR

39 RT-PCR 10 3 Apple chlorotic leaf spot virus (ACLSV) Apple stem pitting virus (ASPV) Apple stem grooving virus (ASGV) reverse transcriptase polymerase chain reaction, RT-PCR Genbank degenerate primer RT-PCR 0.8 agarose gel ASGV 500 bp ASPV 450 bp ACLSV 410 bp RT-PCR ASGV ACLSV ASPV

40 Summary In order to establish a healthy pear seedling production and virus detection system, The growth of two-year-old tissue culture propagated and grafting propagated pear (Pyrus pyrifolia Nakai cv. Henshan) plants was investigated. A RT-PCR assay was used to detect Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV), to test the infection status of pear plants in the production areas. The tissue culture propagated plants grew much faster than the grafting ones. The total leaf fresh weight and dry weight of tissue culture propagated plant was more than 10X of that of grafting propagated plant, the fresh weights and dry weights of shoot and root was more than 3X. The leaves of tissue culture propagated plant had higher chlorophyll content and last longer on the tree than those of grafting propagated plants. Degenerate primers designed from the highly conserved sequences of the above three viruses were used for RT-PCR. Products of 500, 450 and 430 bp for ASGV, ASPV and ACLSV, respectively were routinely detected. In tested fields in production areas, ASGV had the highest infection rate, followed by ACLSV, and then ASPV. All pear production areas in Taiwan should be broadly tested, in order to understand the infection status.

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43 pp Rejuvenation (2) ' ' 55(1) 8-14

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