Research NoteIncubation of plant material with potassium metabisulfite was found to inhibit fungal infections of explants from grapevines. Grapevine tissue of Vitis labruscana cv. Concord was incubated with sulfite pads containing 0.4 g of potassium metabilsufite for one and two days prior to culturing and evaluated against a control that had been surface sterilized with 0.5 % NaOCI and 70 % ethanol after one week for losses due to microbial contamination. Sulfite fumigation of plant material reduced the incidence of mold infection, particularly in tissue cultures developed from fruit explants which had reductions in contamination as high as 10 fold. Continued attempts to isolate contaminants from cultures intitiated from these explants showed no signs of infection

Acree, T. E.

Henick-Kling, T.

Shure, K. B.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 33, 177-178 (1994) Research Note Inhibition of fungal infection using sulfite pads prior to initiation of callus from Vitis labruscana cv. Concord K. B. SHURE, TH. HENICK-KLING and T. E. AcREE S u m m a r y : Incubation of plant material with potassium metabisulfite was found to inhibit fungal infections of explants from grapevines. Grapevine tissue of Vitis labruscana cv. Con-cord was incubated with sulfite pads containing 0.4 g of potas-sium metabilsufite for one and two days prior to culturing and evaluated against a control that had been smface sterilized with 0.5 % NaOCI and 70 % ethanol after one week for Iosses due to microbial contamination. Sulfite fumigation of plant material reduced the incidence of mold infection, particularly in tissue cultures developed from fruit explants which had reductions in contamination as high as 10 fold. Continued attempts to isolate contaminants from cultures intitiated from these explants showed no signs of infection. Introduction : Chemical disinfectants containing NaOCl are commonly used to control bacteria and other microorganisms (T ANNER 1989) including surface sterili-zation of plant material prior to culturing in liquid or solid media (Dooos and RoBERTS 1985). The use of these chemi-eals under heavy fungal pressure can prove inadequate in eliminating mold infection of cultures . Researchprograms involved in tissue culture frequently grow plants in green-houses to reduce Ievels of contamination upon culturing. Preliminary work in the authors Iabaratory with store bought table grapes had low incidence of infection as did cultures prepared with field material early in the season using typical methods of surface sterilization with 0.5 % NaOCl (10 % chlorox) and 70 % ethanol. As the season progressed into the warm humid summer of upstate New York, mold infection of tissue cultures became very fre-quent and attempts to culture material from the field be-came futile . During discussions it was suggested that stor-age with sulfite pads normally used for preservation of grapes during shipping could have been responsible for the Iack of infection seen in the store-bought fmit and might be helpful in reducing infection rates in field material. Sulfite has proven to be an effective inhibitor of yeast (PILKINGTON and RosE 1988) and molds (RAMAKRISHNA et al. 1991). Sulfite fumigation of plant material was tested for its ability to Iimit mold infections in tissue culture. Materials and methods: P l a n t m a t e r i a l : Petioles and immature berries from Concord grape vines were harvested in late August, 1992 from Cornell Vine-yards at the New York State Agricultural Experiment Sta-tion. R e a g e n t s : All media salts, vitamins and growth regulators were purchased from Sigma Chemical Co. Dis-posable, presterilized petri dishes were from Fisher Seien-tific Co. Bacto-agar was purchased from Difco Laborato-ries. The sulfite pads were Uvas Quality Grape Guards manufactured in Chile. These contained approximately 0.40 g of potassium metabisulfite per pad. S u r f a c e s t e r i l i z a t i o n : Explants were washed with moderate agitation in a water/soap solution -about 500 ml distilled or deionized Hp with 5 drops of Micro soap. The tissue was then rinsed thoroughly in dis-tilled water once followed by a 2 min dip in 70 % ethanol. All plant material was then allowed to soak in 10 % chlorox solution for 10 min after which there were 3 rinses in ster-ile, distilled water. All work from the point of chlorox sub-mersion was performed under a laminar flow hood. S u l fit e in c u bat i o n : 15 petioles cut to 10 cm or 25 immature berries still on the duster were placed in a 500 ml beaker containing one sulfite pad at the bottarn and sealed with aluminum foil on the top. Two beakers for each tissue type were prepared. One beaker each from the petioles and from the fruit was incubated for 24 h prior to smface stetilization and the remaining two were incubated for 48 h. All incubations took place at 1 oc. A control group of fruit and petiole tissue was harvested at the same time as the incubated tissue. The control was smface sterilized and cultured without any sulfite incubation within a few hours of harvest from the field . 20 plates were prepared for each treatment and the control. Lass of individual explants and entire petri plates to fungal infections were evaluated after 1 week in culture. Uninfected fruit explants propagated ample callus which was used to initiate cell suspensions in liquid media. These were evaluated every 2 months for contamination on YM and MLB media. C u I t u r i n g o f p 1 a n t m a t e r i a 1 : The petioles had 0.5 cm cut off each end to get rid of disrupted tissue exposed to sterilization chemieals before cutting into 3 mm pieces and plating on media at 5 pieces per plate. The benies were cut into quarters and placed 4 pieces to a plate. Culture media utilized Garnborgs B5 inorganic salts and vitamins (GAMBORG et al. 1968) supplemented with 3 % sucrose, 0.5 mg/1 2,4-D and 0.5 mg/1 BA for the fruit. Media for the petioles was the same except it utilized 0.5 mg/1 IAA as an auxin. 0.8 % Bactoagar was used as a gel-ling agent. Plates were stored in the dark at 23 oc and cal-lus that developed was transferred to new media every 4-6 weeks. Results and discussion: Figure indicates that incuba-tion of fruit explants with sulfite pads for 24 h prior to smface sterilization lowered the incidence of mold infec-tions by 4 fold. Incubating for 48 h cut the infection rate of fruit explants by 10 fold . All uninfected fruit explants ini-tiated callus growth within 3-6 weeks. Callus from the fruit was soft and friable and was off-white to light brown in color. The petiole explants had fewer mold infections due to the treatment, however callus growth was limited. Sus-pensions initiated from fruit callus showed no signs of con-tarri.iriation on YM or MLB media as late as 7 months after sulfite fumigation took place. Cornell University, Food Science and Technology, New York State Agricultural Experiment Station , Geneva, NY 14456, USA 1 7 8  K .  B .  S H u R E ,  T H .  H E N IC K - K L I N G  a n d  T .  E .  A C R E E  S u l f i t e  P a d  l n c u b a t i o n  1 0 0  c :  I  •  F r u i t  E x p l a n t  0  8 0  ü  l ' ? i l  F r u i t  p l a t e  Q )  r m  P e t i o l e  E x p l a n t  -~ . ! :  6 0  ~ P e t i o l e  p l a t e  i U  ~ I  C l  4 0  c :  : : J  u . .  : R .  2 0  0  0  0  2 4  4 8  l n c u b a t l o n  T i m e  F i g u r e :  P e r c e n t  m o l d  i n f e c t i o n  n o t e d  f o r  i n d i v i d u a l  e x p l a n t s  a n d  e n t i r e  p l a t e s  f o r  b o t h  f r u i t  a n d  p e t i o l e  m a t e r i a l  f o r  t h e  c o n t r o l ,  a n d  a f t e r  2 4  a n d  4 8  h  i n c u b a t i o n  p e r i o d s .  T h e  w e t  s u m m e r  s e a s o n  o f  1 9 9 2  c r e a t e d  a m p l e  o p p o r -t u n i t y  f o r  m o l d s  t o  t h r i v e  i n  t h e  v i n e y a r d s  o f  u p s t a t e  N e w  Y o r k .  A s  t h e  s e a s o n  p r o g r e s s e d  i t  b e c a m e  e v i d e n t  t h a t  a c -t i o n  o t h e r  t h a n  s t a n d a r d  c h e m i c a l  s u r f a c e  s t e r i l i z a t i o n  n e e d e d  t o  b e  t a k e n  i f  a n y  e x p l a n t s  p r e p a r e d  w e r e  t o  a v o i d  b r i n g i n g  f u n g a l  g r o w t h  i n t o  c u l t u r e .  T h e  u s e  o f  s u l f i t e  p a d s  f o r  ( s t e r i l i z a t i o n )  f u m i g a t i o n  o f  t h e  p l a n t  m a t e r i a l  a l l o w e d  f o r  c o n t i n u e d  c u l t u r e  o f  m a t e r i a l  f r o m  t h e  f i e l d .  T r e a t m e n t  w i t h  t h e  p a d s  w o r k e d  b e s t  w i t h  f r u i t ,  b u t  a l s o  i n h i b i t e d  i n f e c t i o n  i n  c u l t u r e  w i t h  p e t i o l e  t i s s u e .  O n e  p o s s i b l e  r e a -s o n  f o r  t h e  s l i g h t  d i f f e r e n c e  b e t w e e n  t h e  t w o  t i s s u e s  c o u l d  b e  t h e  c u t t i n g  o f  p e t i o l e  t i s s u e  t h a t  h a d  t o  o c c u r  u p o n  h a r -v e s t  w h e r e a s  t h e  f r u i t  w e n t  i n  t o  i n c u b a t i o n  a s  a  d u s t e r .  E x p o s e d  i n t e r n a l  t i s s u e  c o u l d  h a v e  s e r v e d  a s  r e f u g e  f o r  a n y  m o l d s  o r  m o l d s  s p o r e s  w h i c h  c o n t a m i n a t e d  t h e  p e t i -o l e  a f t e r  c u t t i n g .  T h e  I a c k  o f  c a l l u s  g r o w t h  f r o m  p e t i o l e  m a y  b e  d u e  t o  t h e  d a t e  t h e  m a t e r i a l  w a s  h a r v e s t e d .  C o n t r o l  p l a t e s  t h a t  d i d  r e m a i n  u n i n f e c t e d  a l s o  e x p e r i e n c e d  r e d u c e d  c a l l u s  i n i t i a t i o n .  L a n g e r  i n i t i a t i o n  I i m e s  o r  f a i l u r e s  o f  c a l -l u s  i n i t i a t i n g  w i t h  e x p l a n t s  t a k e n  l a t e  i n  t h e  s e a s o n  h a s  b e e n  n o t e d  p r e v i o u s l y  ( H A W K E R  e t  a l .  1 9 7 3 ;  H o N G  e t  a l .  1 9 8 9 ) .  T h e  b e r r i e s ,  s t i l l  g r e e n  a t  t h e  t i m e  t h i s  m a t e r i a l  w a s  h a r v e s t e d ,  d i d  n o t  i n i t i a t e  c a l l u s  a s  q u i c k l y  a s  f r u i t  p i c k e d  e a r l i e r  i n  t h e  s e a s o n  b u t  a l l  e x p l a n t s  n o t  i n f e c t e d  b y  f u n -g u s  d i d  e v e n t u a l l y  p r o d u c e  a m p l e  c a l l u s .  E x a m i n a t i o n  o f  t h e  r a t e  o f  p l a t e  i n f e c t i o n  ( a t  l e a s t  o n e  e x p l a n t  i n f e c t e d )  s h o w s  t h a t  i n f e c t i o n  o f  t h e  e x p l a n t s  w a s  a p p r o x i m a t e l y  u n i f o r m a l l y  d i s t r i b u t e d  t h r o u g h o u t  t h e  p l a t e s  w i t h i n  e a c h  s e t .  T h i s  d i s t r i b u t i o n  r e s u l t e d  i n  e s s e n t i a l l y  a  c o m p l e t e  l o s s  o f  t h e  c o n t r o l  p l a t e s  f o r  t h e  f r u i t .  T h e  p l a t e s  w i t h  t h e  s u l f i t e  t r e a t e d  e x p l a n t s  t h a t  d i d  n o t  s h o w  i n f e c -t i o n  i n  t h e  i n i t i a l  o n e  w e e k  c u l t u r e  p e r i o d  r e m a i n e d  s t e r i l e  a n d  p r o d u c e d  c a l l u s  w h i c h  i s  s t i l l  b e i n g  p r o p a g a t e d .  R e -p e a t e d  a t t e m p t s  t o  i s o l a t e  c o n t a m i n a n t s  o n  Y M  a n d  M L B  m e d i a  f r o m  s u s p e n s i o n s  i n i t i a t e d  u s i n g  t h i s  c a l l u s  s h o w e d  n o  e v i d e n c e  o f  f u n g i  o r  b a c t e r i a .  T h i s  s t u d y  p r e s e n t s  e v i d e n c e  t h a t  t h e  u s e  o f  s u l f i t e  f u -m i g a t i o n  i s  a  s i m p l e  a n d  e f f e c t i v e  a d j u n c t  t o  t h e  u s e  o f  s o d i u m  h y p o c h l o r i t e  a n d  e t h a n o l  f o r  e x p l a n t  s t e r i l i z a t i o n  a n d  c o u l d  p r o v e  u s e f u l  f o r  r e s e a r c h  p r o g r a m s  t h a t  a r e  l i m -i t e d  t o  f i e l d  m a t e r i a l  i n  h u m i d  c l i m a t e s .  A c k n o w l e d g e m e n t s :  T h e  a u t h o r s  w o u l d  l i k e  t o  t h a n k  V e n -t u r e  V i n e y a r d s  o f  L o d i  N Y  f o r  d o a n t i n g  t h e  s u l f i t e  p a d s  a n d  t o  B .  I .  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Inhibition of fungal infection using sulfite pads prior to initiation of callus from Vitis labruscana  cv. Concord

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Inhibition of fungal infection using sulfite pads prior to initiation of callus from Vitis labruscana cv. Concord

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