Biotechnology for Crop Protection - American Chemical Society

Chapter

Immunochemical Propagative and

1

Biotechnology for Crop Protection Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.

Technology

Plant

Bacteria

26

in

Parts the

in for

Indexing Viruses

Netherlands

1

2

J. W. L. van Vuurde , D. Z. Maat , and A. A. J. M. Franken 1

Research Institute for Plant Protection, P.O. Box 9060, 6700 GW Wageningen, Netherlands Government Seed Testing Station, P.O. Box 9104, 6700 HE Wageningen, Netherlands Immunological methods, mainly ELISA, were used in the Netherlands during 1986 for c. 3.2 x 10 tests for quality indexing propagation material for viruses, viz: seed potatoes (46%), ornamentals (30%), bulbs (17%), vegetable seeds (3.5%) and fruit trees (3.5%). Tests are mostly done by inspection services under the supervision of the Ministry of Agriculture (78%). Indexing for plant pathogenic bacteria was done with ELISA for blackleg in seed potatoes (2.5 x 10 tests) and with immunofluorescence microscopy (IF) for bacteria in seeds and potted plants. Immuno-isolation and immunofluorescence colony staining, techniques which combine the advantages of serology and agar plating, show good prospects to overcome problems in the sensitivity and reliability of detection methods for plant pathogenic bacteria. 2

6

5

Reliable and quick detection methods which can be applied to large series of samples at low costs are important tools for quality control of agricultural products and for ecological and environmental studies. Indexing of plant propagation materials for pathogens is an important aspect of the production of high-quality plant products in the Netherlands. This approach has been of great importance in restricting the economic damage caused by viruses and bacteria, pathogens which generally cannot be killed effectively in plants by chemicals or any other method. Quality indexing has also improved the marketing value of export plant propagation materials. Laboratory indexing in addition to field inspection will give extra guarantees with regard to propagative material contaminated with economically important pathogens and organisms mentioned in quarantine lists of importing countries (4). Serological techniques, such as latex agglutination and micro-precipitation, which were used for routine virus indexing in c

0097-6156/88/0379-0338$06.00/0 1988 American Chemical Society


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VAN VUURDE E T A L .

Indexing Propagative Plant Parts

339

the N e t h e r l a n d s have been r e p l a c e d s u c c e s s f u l l y by E L I S A . The development o f s e n s i t i v e and r e l i a b l e s e r o l o g i c a l methods f o r f u n g i , nematodes, i n s e c t s and b a c t e r i a i s much more c o m p l i c a t e d t h a n f o r v i r u s e s . T h i s i s due t o the presence o f many n o n - t y p i c a l a n t i g e n i c s t r u c t u r e s i n t h e s e organisms w h i c h a l s o can be found i n n o n p a t h o g e n i c organisms and l e a d t o f a l s e p o s i t i v e r e a c t i o n s . I n s p i t e of t h e s e l i m i t a t i o n s , r e a s o n a b l y r e l i a b l e a s s a y s were developed t o d e t e c t some e c o n o m i c a l l y i m p o r t a n t b a c t e r i a i n seed l o t s ( 1 8 , 3 1 ) . The aim o f t h i s paper i s t o p r e s e n t the commonly used ELISA p r o c e d u r e s f o r i n d e x i n g o f p l a n t pathogens and t h e i r a p p l i c a t i o n i n the N e t h e r l a n d s . The schemes d e v e l o p e d f o r the d e t e c t i o n o f seed-borne v i r u s e s a r e used t o i l l u s t r a t e how problems i n the r o u t i n e procedures can be s o l v e d . I n a d d i t i o n , ways t o overcome problems i n ELISA f o r o t h e r t y p e s o f p l a n t m a t e r i a l and the p o s s i b i l i t i e s f o r automating ELISA a r e d e s c r i b e d . F i n a l l y , the p r e s e n t s t a t u s and s t r a t e g i e s f o r i m p r o v i n g the d e t e c t i o n o f p l a n t pathogenic b a c t e r i a are presented. A p p l i c a t i o n o f ELISA i n P l a n t P a t h o l o g y S i n c e i t s i n t r o d u c t i o n i n m e d i c a l r e s e a r c h i n 1971, v a r i o u s types o f enzyme immunoassays have been d e v e l o p e d ( 2 1 ) . The double a n t i b o d y sandwich ELISA (DAS-ELISA) i n t r o d u c e d i n p l a n t p a t h o l o g y i n 1976 (23) has become the major t e s t system f o r p l a n t i n d e x i n g i n the N e t h e r l a n d s . T h i s t e c h n i q u e can be a p p l i e d t o a wide range o f p l a n t p a t h o g e n s . F i g u r e 1 shows the p r i n c i p l e f o r the complex a n t i g e n s i t u a t i o n for detecting bacteria (25). The major advantages o f c o a t i n g the s o l i d phase w i t h a n t i b o d i e s over the d i r e c t c o a t i n g o f the t e s t sample onto the s o l i d phase a r e a s e l e c t i v e c o n c e n t r a t i o n o f the a n t i g e n from the sample onto the s o l i d phase and the washing away o f the unbound sample m a t e r i a l . Sample compounds bound t o t h e s o l i d phase may i n c r e a s e the background r e a c t i o n . V i r u s p a r t i c l e s a r e s t r o n g l y bound to the s o l i d phase by the c o a t i n g a n t i b o d i e s , but m i c r o s c o p i c a l i n v e s t i g a t i o n s a t the R e s e a r c h I n s t i t u t e f o r P l a n t P r o t e c t i o n has shown t h a t the much l a r g e r b a c t e r i a l c e l l s a r e washed out o f the w e l l s . Only s m a l l p a r t i c l e s such as f l a g e l l a e , c e l l w a l l p a r t i c l e s and s o l u b l e a n t i g e n s a r e i n v o l v e d i n the r e a c t i o n . Enzymes l i k e a l k a l i n e phosphatase have l i n e a r r e a c t i o n k i n e t i c s , r e s u l t i n g i n a b e t t e r d i s c r i m i n a t i o n between a n t i g e n - c o n t a i n i n g and a n t i g e n - f r e e samples when the background r e a c t i o n can be kept s u f f i c i e n t l y low ( 5 ) . V i r u s d e t e c t i o n w i t h the commonly used immunoglobulin G (IgG) type a n t i - v i r u s a n t i b o d i e s (MW c . 150,000) conjugated w i t h enzyme ( e . g . a l k a l i n e phosphatase, MW 8 0 , 0 0 0 - 1 0 0 , 0 0 0 ) i s more s p e c i f i c than the i n d i r e c t p r o c e d u r e u s i n g non-conjugated a n t i - v i r u s IgG and a l k a l i n e p h o s p h a t a s e - c o n j u g a t e d second a n t i b o d i e s ( 1 2 , 1 3 ) . T h i s i s e x p l a i n e d from s t e r i c h i n d r a n c e o f the a n t i b o d y by the enzyme m o l e c u l e s w h i c h reduces the p o s s i b i l i t i e s f o r c o u p l i n g t o p a r t l y homologous a n t i g e n i c d e t e r m i n a n t s . The b e s t a n t i b o d y s p e c i f i c i t y can be e x p e c t e d from t e s t systems u s i n g q u a l i f i e d m o n o c l o n a l a n t i b o d i e s . However, i n i n d e x i n g p l a n t m a t e r i a l f o r v i r u s e s i n the N e t h e r l a n d s , none o f the m o n o c l o n a l a n t i b o d i e s o b t a i n e d from v a r i o u s o r i g i n s gave b e t t e r o r even e q u a l l y good r e s u l t s so f a r when compared t o t e s t i n g with polyclonal antisera.


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BIOTECHNOLOGY FOR CROP PROTECTION

5

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Figure 1. Double antibody sandwich ELISA in a well of a microtitre plate (W, washing with PBS-Tween). 1. Solid-phase coating step: A , incubation with antibodies in high p H buffer, B, solid-phase bound antibodies. 2. Sample step: A , sample incubation in PBS-Tween; B, detail showing antigens trapped by homologous antibodies. 3. Enzyme conjugate step: binding of conjugate molecules to homologous antigens from the sample. 4. Substrate step: color change of the substrate due to enzyme activity of the antigen-bound enzyme conjugate in the case of a positive reaction. 5. Diagram of a bacterium showing various sources of antigen. (Reproduced with permission from ref. 25. Copyright 1988 Akademiai Kiado.)


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ELISA i n R o u t i n e I n d e x i n g * T a b l e I summarizes the major types o f p l a n t m a t e r i a l s and pathogens f o r w h i c h r o u t i n e q u a l i t y i n d e x i n g w i t h ELISA i s done i n the N e t h e r l a n d s i n 1986. C i r c a 3 . 2 x 1 0 ° t e s t s a r e done f o r the d e t e c t i o n o f v i r u s e s , m o s t l y i n seed p o t a t o e s , o r n a m e n t a l s , and f l o w e r b u l b s . The v a r i o u s v i r u s e s i n v o l v e d i n seed p o t a t o i n d e x i n g comprise 46% o f t h e s e . I n T a b l e I I the v i r u s e s f o r seed p o t a t o e s a r e s p e c i f i e d . F o r b a c t e r i a , 0.25 x 10 t e s t s were done, and o n l y f o r seed p o t a t o e s . The p o t a t o b l a c k l e g organism ( E r w i n i a c a r o t o v p r a s u b s p . a t r o s e p t i c a ) , i s the o n l y b a c t e r i a l pathogen f o r w h i c h ELISA i s a p p l i e d on a l a r g e s c a l e . A p p l i c a t i o n o f E L I S A , however, has many l i m i t a t i o n s f o r i t s use i n p h y t o b a c t e r i o l o g y compared t o o t h e r s e r o l o g i c a l t e c h n i q u e s l i k e immunof l u o r e s c e n c e m i c r o s c o p y and t r a d i t i o n a l p l a t i n g t e c h n i q u e s ( 3 2 ) .

Table I .

I n d e x i n g p r o p a g a t i o n m a t e r i a l w i t h ELISA i n the N e t h e r l a n d s i n 1986

Crop

Pathogen

T e s t s per y e a r *

Percentage

Seed p o t a t o e s

Viruses Bacteria Viruses Viruses Viruses Viruses

1,465,000 250,000 550,000 965,000 110,000 110,000

42.5 7.2 15.9 28.0 3.2 3.2

Bulbs Ornamentals V e g e t a b l e seeds Fruit trees

T o t a l number o f t e s t s

3,450,000

F i g u r e s a r e e s t i m a t e s based on the amounts o f a n t i s e r a

Table I I .

I n d e x i n g seed p o t a t o e s f o r v i r u s e s and b a c t e r i a i n 1986

Pathogen

T e s t s per y e a r *

Potato l e a f r o l l virus A " M

M

M

supplied.

virus

s X Y

T o t a l number: Virus tests Bacterial tests (blackleg)

565,666 305,000 10,000 310,000 325,000 310,000

1,465,000 250,000

Percentage 14.0 20.8 0.6 21.2 22.2 21.2

100.0

F i g u r e s a r e e s t i m a t e s based on the amounts o f a n t i s e r a

supplied.

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ELISA f o r t h e , D e t e c t I o n o f Seed-borne V i r u s e s Many a s p e c t s i n v o l v e d i n ELISA f o r i n d e x i n g p l a n t m a t e r i a l s can be demonstrated from the p r o c e d u r e s f o r seed t e s t i n g . F o r r e l i a b l e i n d e x i n g , p r o p e r s a m p l i n g procedures a r e d e s c r i b e d i n the handbook o f the I n t e r n a t i o n a l Seed T e s t i n g A s s o c i a t i o n ( 1 0 ) . F i g u r e 2 p r e s e n t s two schemes w h i c h a r e developed a t the Government Seed T e s t i n g S t a t i o n f o r seed t e s t i n g on the base o f e x p e r i e n c e w i t h v a r i o u s types o f s e e d s . The f i r s t scheme d e a l s w i t h s m a l l seeds w h i c h can be e a s i l y g e r m i n a t e d i n l a r g e q u a n t i t i e s i n a l i m i t e d space a t s t a n d a r d c o n d i t i o n s adapted from the ISTA handbook ( 1 0 ) . The second scheme d e a l s w i t h l a r g e r t y p e s , l i k e pea and bean s e e d , f o r w h i c h g e r m i n a t i o n i s much more l a b o r i o u s and c r i t i c a l . F o r t h e s e s e e d s , d r y h o m o g e n i z a t i o n has shown t o be most e f f i c i e n t . The f o l l o w i n g examples i l l u s t r a t e the procedures and problems i n t e s t i n g s m a l l seeds and l a r g e r seeds• S m a l l Seeds. I n the N e t h e r l a n d s , l e t t u c e seed l o t s a r e c e r t i f i e d w i t h r e g a r d t o l e t t u c e mosaic v i r u s when 2,000 seeds a r e found f r e e of the a g e n t . ELISA was developed t o overcome the sometimes i n s u f f i c i e n t r e l i a b i l i t y and the h i g h c o s t s o f the g r o w i n g - o n t e s t (17) and the Chenopodium t e s t (15) used e a r l i e r . Comparison o f the r e s u l t s o f the g r o w i n g - o n t e s t performed under optimum c o n d i t i o n s w i t h those o f E L I S A , u s i n g subsamples c o n t a i n i n g i n c r e a s i n g numbers o f s e e d s , demonstrated t h a t even one s l i g h t l y i n f e c t e d seed i n 99 h e a l t h y seeds c o u l d be r e l i a b l y d e t e c t e d w i t h ELISA ( 2 8 ) . T h e r e f o r e , o f a seed l o t 20 subsamples o f 100 seeds each a r e t e s t e d i n E L I S A . The subsamples a r e i n c u b a t e d f o r 4 days a t 20°C i n a g e r m i n a t i o n box (20x15x2.5 cm) w i t h p l e a t e d wet f i l t e r p a p e r . One subsample i s s p r e a d out i n one p l e a t , the maximum c a p a c i t y per box i s 40 s u b s a m p l e s . P r e c h i l l i n g f o r 2 days a t 10°C i s used t o b r e a k dormancy i n the seeds and t o o p t i m i z e u n i f o r m g e r m i n a t i o n . A t the end o f the g e r m i n a t i o n p e r i o d the r o o t s o f the s e e d l i n g s a r e grown t o g e t h e r and form a s t r i p w h i c h can be e a s i l y t a k e n o f f the f i l t e r p a p e r . The e x t r a c t i o n o f the subsample can be done i n about 15 seconds by g r i n d i n g the s t r i p w i t h a power d r i v e n c r u s h e r ( P o l l & h n e , F R G ) . D u r i n g p r e s s i n g , 0 . 5 m l o f e x t r a c t i o n b u f f e r i s added t o the r o l l e r s o f the p r e s s and c 0.8 ml o f e x t r a c t i s c o l l e c t e d i n a 1 ml p o l y p r o p y l e n e t e s t t u b e . The 20 subsample tubes a r e p l a c e d i n a tube r a c k adapted t o the use o f an e i g h t - c h a n n e l p i p e t f o r r o u t i n e l o a d i n g o f m i c r o t i t r e p l a t e s . The P o l l & h n e c r u s h e r c o n t a i n s a v i g o r o u s washing system to c l e a n the p r e s s i n between subsamples i n s i x s e c o n d s . The DAS-ELISA i s performed a c c o r d i n g t o C l a r k and Adams ( 6 ) , but 0.05 M phosphate i s used i n the b u f f e r f o r the e x t r a c t i o n o f the p l a n t sample and f o r the d i l u t i o n o f the a l k a l i n e phosphatase c o n j u g a t e . The o p t i c a l d e n s i t y (0D) o f the s u b s t r a t e s i s measured w i t h a photometer a t 405 nm. As shown i n F i g u r e 2 , the r e s u l t s f o r the t e s t sample a r e r e p o r t e d n e g a t i v e i f a l l subsamples a r e n e g a t i v e . The percentage o f i n f e c t e d seeds i n a p o s i t i v e sample can be e s t i m a t e d w i t h a s t a t i s t i c a l t a b l e from the number o f p o s i t i v e subsamples out o f the 20 subsamples ( 2 8 ) . T h i s t e s t w i l l d e t e c t p r e d o m i n a n t l y s e e d - t r a n s m i t t e d v i r u s as i t i s c a r r i e d out on germinated s e e d s . When d r y seeds a r e u s e d , the e x t r a c t i o n o f the seeds i s l e s s c o n s i s t e n t , more l a b o r i o u s and f a l s e p o s i t i v e r e s u l t s may be o b t a i n e d w i t h r e g a r d t o a c t u a l seed t r a n s m i s s i o n because

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SAMPLE

SMALL SEEDS

LARGE SEEDS

e.g. LMV in lettuce seed Germinate Press

e.g. PEBV & PSbMV in pea seed

20 subs.of 100 seeds 20 subs, of y 100 seedlings

Dry

1,600 seeds

Grind

16 subs, of 100 dry seeds

I S E> M Germination *S ELISA (16x100) ^ MPN I Report negative — —

Report % seedtransmission

Report negative

Standard procedure Optional confirmation procedure

Report % seed-borne

Y

Report % seedtransmission

F i g u r e 2 . G e n e r a l s t r a t e g i e s f o r ELISA r o u t i n e t e s t i n g o f seeds f o r v i r u s , i l l u s t r a t e d f o r l e t t u c e mosaic v i r u s (LMV) i n l e t t u c e and pea e a r l y - b r o w n i n g v i r u s (PEBV) and pea seed-borne mosaic v i r u s (PSbMV) i n p e a . Schemes i n c l u d e t h e e s t i m a t i o n o f t h e p e r c e n t a g e o f i n f e c t e d seeds w i t h t h e most p r o b a b l e number (MPN) method, c o n f i r m a t i o n o f d o u b t f u l r e s u l t s by immunosorbent e l e c t r o n m i c r o s c o p y (ISEM) and check on a c t u a l seed t r a n s m i s s i o n of the v i r u s to s e e d l i n g s .

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seed-borne v i r u s from non-embryonic p a r t s o f the ungerminated seed may be i n v o l v e d i n the r e a c t i o n ( 4 ) . L a r g e Seeds. The d i f f i c u l t h a n d l i n g and the more c r i t i c a l g e r m i n a t i o n o f pea seeds has l e d to a t e s t system based on the g r i n d i n g o f subsamples o f 10 t o 100 seeds i n t o a f i n e f l o u r (29)* T h i s method i s used t o e l i m i n a t e the m a j o r i t y o f the n e g a t i v e samples i n the most e f f i c i e n t way* The t e s t i n g scheme f o r pea seed-borne mosaic v i r u s and f o r pea e a r l y - b r o w n i n g v i r u s i s g i v e n i n F i g u r e 2* I n a seed l o t , 16 subsamples o f 100 seeds a r e prepared u s i n g semi-automated equipment* Each subsample i s ground s e p a r a t e l y i n a heavy c o f f e e m i l l w i t h exchangeable g r i n d e r p a r t * A subsample i s ground i n t o a f i n e f l o u r i n about 30 seconds* A one ml s y r i n g e w i t h a c u t t i p i s used t o sample c* 0*20 g o f f l o u r from the g r i n d i n g head by v e r t i c a l p r e s s i n g the c u t s y r i n g e i n t o the f l o u r , w i t h the p l u n g e r f i x e d i n the r i g h t p o s i t i o n , u n t i l the c a v i t y i s f i l l e d and t o t r a n s f e r the s y r i n g e i n t o a 1 m l p o l y p r o p y l e n e t u b e . A f t e r a d d i t i o n o f c . 0.7 m l o f 0*05 M phosphate e x t r a c t i o n b u f f e r w i t h 0*15% Tween 20 and thorough m i x i n g , the same ELISA procedures as used f o r s m a l l seeds a r e f o l l o w e d * Between a s e r i e s o f subsamples the g r i n d i n g p a r t s a r e c l e a n e d * The s t a n d a r d t e c h n i q u e can be improved however by c o m b i n i n g sample and c o n j u g a t e i n c u b a t i o n * T h i s m o d i f i c a t i o n n o t o n l y reduces the number o f s t e p s i n the t e s t , but a l s o i n c r e a s e s the d i f f e r e n c e s between h e a l t h y and v i r u s - c o n t a i n i n g samples by r e d u c i n g the background r e a c t i o n (29)* P o s i t i v e samples i n t h i s s c r e e n i n g can be f u r t h e r i n v e s t i g a t e d f o r seed t r a n s m i s s i o n by t e s t i n g 1,600 s e e d l i n g s i n ELISA* The s t r u c t u r e and the h u m i d i t y o f the s o i l a r e v e r y c r i t i c a l t o o b t a i n c o n s i s t e n t g e r m i n a t i o n * The s e e d l i n g s a r e e x t r a c t e d i n subsamples o f 100 w i t h the P o l l & h n e c r u s h e r and the sap d i l u t e d i n e x t r a c t i o n b u f f e r ( 1 : 5 ) and t e s t e d as d e s c r i b e d f o r l e t t u c e mosaic v i r u s * Pea seed-borne mosaic v i r u s almost has the s t a t u s o f a q u a r a n t i n e d i s e a s e and a l l p o s i t i v e samples i n the ELISA s c r e e n i n g a r e r e j e c t e d as b e i n g p o t e n t i a l l y dangerous* I n case f l o u r t e s t i n g g i v e s a v e r y weak r e a c t i o n i n E L I S A , c o n f i r m a t i o n i s needed e s p e c i a l l y f o r q u a r a n t i n e type d i s e a s e s l i k e pea seed-borne mosaic v i r u s * Immunosorbent e l e c t r o n m i c r o s c o p y p r o v e d t o be a v e r y s e n s i t i v e t o o l f o r c o n f i r m a t i o n and e n a b l e s t o use the f l o u r o f the t e s t sample t h a t gave a d o u b t f u l ELISA r e s u l t * The model f o r l a r g e seeds p r e s e n t e d i n F i g u r e 2 was a l s o s u c c e s s f u l a t the Government Seed T e s t i n g S t a t i o n t o d e t e c t squash mosaic v i r u s i n melon seeds* ELISA f o r V i r u s e s i n V e g e t a t i v e P l a n t M a t e r i a l s I n a d d i t i o n t o s e e d s , o t h e r p l a n t p a r t s as l e a v e s , stems, b a r k , r o o t s , b u l b s , and t u b e r s a r e t e s t e d * The ELISA p r o c e d u r e s used f o r the v a r i o u s p r o d u c t s w i l l be m a i n l y the same* However, sample p r e p a r a t i o n v a r i e s w i d e l y among p l a n t s p e c i e s and p l a n t p a r t s * I n the N e t h e r l a n d s , the P o l l f l h n e p r e s s i s the most p o p u l a r t o o l t o p r e p a r e e x t r a c t s * R e l a t i v e l y d r y p l a n t p a r t s such as the l e a v e s o f some f r u i t t r e e s , b a r k m a t e r i a l , r o o t s , b u l b s and t u b e r s a r e e x t r a c t e d by a d d i n g b u f f e r t o the p r e s s j u s t above the sample d u r i n g grinding. P l a n t m a t e r i a l s which g i v e very s l i m y e x t r a c t s , are p r o p e r l y d i l u t e d t o o b t a i n good r e s u l t s . I n case o f l i l y b u l b s c a l e s , the


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d e t e c t i o n o f v i r u s e s i s enhanced r e m a r k a b l y when the e x t r a c t s a r e i n c u b a t e d w i t h enzymes ( e s p e c i a l l y h e m l c e l l u l a s e , 2 ) . N o n - s p e c i f i c r e a c t i o n s i n ELISA may be caused by a n t i b o d i e s b i n d i n g t o normal p l a n t a n t i g e n s , o r by n o n - i m m u n o l o g i c a l b i n d i n g o f enzyme. The l a t t e r can o f t e n be i n h i b i t e d by proper sample d i l u t i o n a n d / o r by a d d i n g r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f Tween 20 (up t o 2%), p o l y v i n y l p y r r o l i d o n (up t o 2%), c r u d e egg a l b u m i n ( 0 . 1 - 4%) o r skimmed m i l k powder (3%), o r c o m b i n a t i o n s o f t h e s e compounds t o the e x t r a c t i o n a n d / o r c o n j u g a t e b u f f e r . A l s o r e d u c i n g a g e n t s a r e sometimes a p p l i e d , e . g . sodium s u l p h i t e , 0.01 - 0 . 0 2 m o l / 1 , i s used i n b u f f e r s f o r the t e s t i n g o f l e t t u c e , p e l a r g o n i u m , and chrysanthemum. When t e s t i n g f l o w e r b u l b m a t e r i a l s , n o n - s p e c i f i c r e a c t i o n s a r e reduced by i n c r e a s i n g the Tween c o n c e n t r a t i o n and by a d d i n g 1% normal h o r s e serum t o the e x t r a c t i o n b u f f e r ( 2 0 ) . Egg a l b u m i n o r m i l k powder p r o v e d h e l p f u l i n r e d u c i n g background r e a c t i o n s o b t a i n e d w i t h g a r l i c and s h a l l o t ( 7 ) , whereas egg a l b u m i n (1%) t o g e t h e r w i t h an i n c r e a s e d Tween c o n c e n t r a t i o n (1%) were n e c e s s a r y t o r e l i a b l y t e s t l e e k (Research I n s t i t u t e f o r P l a n t P r o t e c t i o n ) . The r e l i a b i l i t y o f the t e s t i s I n f l u e n c e d by the s e a s o n , the p l a n t p a r t t e s t e d and i t s p h y s i o l o g i c a l s t a t e . T h e r e f o r e , c e r t a i n t e s t s cannot be done r e l i a b l y i n summer ( e . g . c a r n a t i o n s f o r c a r n a t i o n e t c h e d r i n g v i r u s ) . I n dormant p o t a t o t u b e r s , f o r some viruses r e l i a b l e detection i s possible only after breaking of dormancy ( e . g . by r i n d i t e t r e a t m e n t , 9 ) . V i r u s e s a l s o a r e not always e v e n l y spread throughout the p l a n t , r e q u i r i n g c a r e f u l s a m p l i n g . Equipment f o r R o u t i n e H a n d l i n g and A u t o m a t i o n o f ELISA To f a c i l i t a t e and speed up ELISA p r o c e d u r e s , b e s i d e s the P o l l S h n e p r e s s , 8 - c h a n n e l p i p e t s and s p e c i a l tube r a c k s mentioned a l r e a d y ( F i g u r e 3 ) , o t h e r equipment i s used o r under development. Based on c o m m e r c i a l l y a v a i l a b l e m u l t i c h a n n e l pumps, equipment has been d e v e l o p e d t o f i l l s e r i e s o f m i c r o t i t r e p l a t e s w i t h c o a t i n g s o l u t i o n s , enzyme c o n j u g a t e s o r s u b s t r a t e s . As i t i s o f t e n d i f f i c u l t to c o m p l e t e l y remove p l a n t e x t r a c t s from the w e l l s , h i g h - p r e s s u r e washing d e v i c e s have been c o n t r u c t e d . I n many systems r e s u l t s a r e read w i t h the nake e y e . However, more and more t h i s i s r e p l a c e d by ( s e m i - ) a u t o m a t i c r e a d i n g and r e c o r d i n g , f o r i n s t a n c e d i r e c t l y on sample a t t e s t a t i o n f o r m s , by c o m p u t e r i z e d p h o t o m e t e r s . Most h a n d l i n g s a r e f a i r l y e a s i l y mechanized o r automated. However, the p r e p a r a t i o n o f the sample demands an i n d i v i d u a l approach depending on the t y p e o f p l a n t m a t e r i a l t o be t e s t e d . Major p r o g r e s s i n t h i s r e s p e c t has been made f o r the a u t o m a t i c sample h a n d l i n g o f b u l b s c a l e s ( 2 0 ) . The scheme o f t h i s procedure i s g i v e n i n Figure 4. Indexing f o r P l a n t Pathogenic, B a c t e r i a B e s i d e s t h e i r s u i t a b i l i t y f o r r o u t i n e a p p l i c a t i o n , immunof l u o r e s c e n c e ( I F ) and e s p e c i a l l y ELISA have some s t r o n g l i m i t a t i o n s f o r d e t e c t i n g b a c t e r i a ( 3 1 , 3 2 ) . The d e t e c t i o n l i m i t o f IF i s 1 0 t o 10 c e l l s per m l , whereas ELISA needs even 10 t o 100 t i m e s more c e l l s per m l ( 1 , 2 6 ) . F u r t h e r m o r e , f a l s e p o s i t i v e r e s u l t s may be o b t a i n e d when s a p r o p h y t i c m i c r o - o r g a n i s m s w i t h s i m i l a r a n t i g e n i c d e t e r m i n a n t s a r e p r e s e n t i n the t e s t s a m p l e s . Compared t o J


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Figure 3. Equipment for routine application of ELISA: extraction of plant material with a Pollahne press and transfer of samples from tubes to corresponding wells in an ELISA microtitre plate with an 8-channel pipet.

1 TT

2

3

U

5

6

Figure 4. Scheme of the Berleco automated extraction and microtitre plate loading equipment. 1. Filling plastic bag from a belt with plant material (bulb scale) and buffer. 2. Sealing. 3. Pressing. 4. Inversion of the belt. 5. Sampling with syringe through valve. 6. Loading well in microtitre plate. (Reproduced with permission from ref. 20. Copyright 1984 Hofetad Vakpers bv.)

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t r a d i t i o n a l i s o l a t i o n methods, t h e advantages o f I F and ELISA a r e t h e i r c o n s i s t e n c y , s e n s i t i v i t y , speed and s u i t a b i l i t y f o r r o u t i n e a p p l i c a t i o n . The u s e o f a n t i s e r a , w h i c h a r e checked f o r s p e c i f i c i t y w i t h t h e a i d o f s e l e c t e d s t r a i n s o f s a p r o p h y t e s i s o l a t e d from t h e type o f p l a n t m a t e r i a l t o be t e s t e d , l i m i t s t h i s r i s k on f a l s e p o s i t i v e r e a c t i o n s ( 2 2 ) . I f t h e i n t e r f e r e n c e o f s a p r o p h y t e s can be l i m i t e d , i s o l a t i o n on a s u i t a b l e medium i s an Important t o o l t o confirm a s e r o l o g i c a l p o s i t i v e sample. Seed T e s t i n g w i t h I F . The f o l l o w i n g procedure f o r t h e d e t e c t i o n o f Pseudomonas s y r i n g a e p v . p h a s e o l i c o l a i n bean seed was s a t i s f a c t o r y f o r r o u t i n e i n d e x i n g and nas t e e n used a t t h e Government Seed T e s t i n g S t a t i o n i n t h e N e t h e r l a n d s s i n c e 1982 ( 2 6 ) . The t e s t sample i s d i v i d e d i n t o f i v e subsamples o f 1,000 s e e d s . The subsamples a r e soaked i n s t e r i l e water i n a p l a s t i c bag a t 6 °C f o r 24 h . A 1 m l sample o f t h e s o a k i n g s o l u t i o n i s t a k e n from each subsample a f t e r 6 h t o p r e p a r e I F s l i d e s and t h e r e m a i n i n g p a r t i s s t o r e d a t - 1 8 ° C . A sample w i t h f i v e I F n e g a t i v e subsamples i s r e p o r t e d ' n e g a t i v e ' (pathogen f r e e o r l e s s than 10 c e l l s / m l ) . F o r I F p o s i t i v e subsamples, a g a i n 1 m l s o a k i n g s o l v e n t i s t a k e n 24 h a f t e r s t a r t i n g the i n c u b a t i o n and a n a l y z e d by I F and p l a t i n g on K i n g ' s medium B . C o l o n i e s p r o d u c i n g a b l u e f l u o r e s c e n t pigment under U V - l i g h t a r e b i o c h e m i c a l l y t e s t e d and i n v e s t i g a t e d f o r p a t h o g e n i c i t y on bean s e e d l i n g s . Samples w i t h one o r more p o s i t i v e subsamples i n I F and from w h i c h t h e pathogen c o u l d be i s o l a t e d a r e r e p o r t e d as P . s . p v . p h a s e o l i c o l a c o n t a m i n a t e d . Samples p o s i t i v e by I F and n e g a t i v e by p l a t i n g a r e r e p o r t e d as p o t e n t i a l l y dangerous when s a p r o p h y t i c bacteria interfered seriously i n plating. S i m i l a r p r o c e d u r e s a r e worked o u t f o r Xanthomonas c a m p e s t r i s p v . c a m p e s t r i s i n cabbage seeds ( 1 9 ) X . c . p v . p h a s e o l i ( 1 4 , 2 6 ) , P . s . p v . p l s i * T G o v e r n m e n t Seed T e s t i n g S t a t i o n ) and f o r C l a y i b a c t e r m i c h l g a n e n s i s s u b s p . sepedonicus f o r t e s t i n g seed p o t a t o e s i n t h e European Community ( i f , 16) • T e s t i n g Seed P o t a t o Tubers w i t h E L I S A . ELISA was f i r s t r e p o r t e d f o r d e t e c t i n g E r w i n i a c a r o t o v o r a s u b s p . a t r o s e p t l c a ( b l a c k l e g organism) i n seed p o t a t o e s (£4]). S t a r t i n g i n l ^ t o w i t h l a r g e s c a l e I n d e x i n g , 2 . 5 x 10 t e s t s a r e now done p e r y e a r f o r seed p o t a t o e s ( T a b l e I I ) by t h e G e n e r a l N e t h e r l a n d s I n s p e c t i o n S e r v i c e f o r A g r i c u l t u r a l Seeds and Seed P o t a t o e s ( N A K ) . S i n c e 1987, seed p o t a t o e s a r e s i m u l t a n e o u s l y t e s t e d f o r E . c h r y s a n t h e m i . Reasonable c o r r e l a t i o n s were o b t a i n e d between l a f t e s t i n g / o r E_.c. s u b s p . a t r o s e p t l c a (50 t u b e r s ) and symptom development i n t h e NAK c o n t r o l f i e l d (400 p l a n t s ) i n s p i t e o f t h e l i m i t a t i o n s o f ELISA ( 8 ) . New Approach i n Deitecting Bac t e r i a The s e n s i t i v i t y o f s e r o l o g i c a l t e s t s i s n o t i n f l u e n c e d by t h e r e l a t i v e I n c i d e n c e o f s a p r o p h y t e s i n t h e sample u n l e s s they r e a c t w i t h t h e a n t i s e r u m . Major c r i t i c a l parameters o f ELISA and I F f o r r e l i a b l e r i s k assessment o f p l a n t m a t e r i a l a r e t h e s p e c i f i c i t y o f the s e r o l o g i c a l r e a c t i o n and t h e s t a t u s o f t h e b a c t e r i a l c e l l s , v i z . v i a b l e c e l l s , dead c e l l s a n d / o r c e l l components o f t h e homologous o r g a n i s m . I s o l a t i o n o f t h e t a r g e t organism i n p r i n c i p l e e n a b l e s a 100% r e l i a b l e i d e n t i f i c a t i o n , an 1 , 0 0 0 : 1 . I s o l a t i o n o f b a c t e r i a from I F - p o s i t i v e c o l o n i e s c a n be done w i t h a f i n e n e e d l e o r g l a s s c a p i l l a r y . The method w i l l be e v a l u a t e d f o r other plant pathogenic b a c t e r i a . Optimum s e n s i t i v i t y and s p e c i f i c i t y c a n be o b t a i n e d when i m m u n o - i s o l a t i o n and immunofluorescence c o l o n y s t a i n i n g a r e a p p l i e d i n c o m b i n a t i o n ( 3 2 ) . Immunofluorescence c o l o n y s t a i n i n g can be adapted i n t o r o u t i n e p r o c e d u r e s , e . g . , by u s i n g w e l l s o f t i s s u e c u l t u r e p l a t e s t o r e p l a c e p e t r i d i s h e s . Reading can be automated u s i n g m i c r o p r o c e s s o r c o n t r o l l e d v i d e o s c a n n i n g equipment. S t r a t e g i e s f o r Improvement, o f A n t i s e r u m S p e c i f i c i t y f o r B a c t e r i a . flie p o s s i b i l l t y or i s o l a t i n g b a c t e r i a from I F - s t a i n e c l c o l o n i e s ' p r o v i d e s a v e r y e f f i c i e n t t o o l f o r i n v e s t i g a t i n g the r i s k on f a l s e p o s i t i v e r e a c t i n g m i c r o - o r g a n i s m s . T h i s i s done by t e s t i n g h e a l t h y m a t e r i a l o f v a r i o u s o r i g i n s f o r the p r e s e n c e o f b a c t e r i a w h i c h form c o l o n i e s that c r o s s - r e a c t i n I F . I s o l a t i o n of these c r o s s - r e a c t i n g

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strains is of major importance to identify typical antigens in the target bacteria. The knowledge of these antigens will improve the production of more specific polyclonal antisera and of monoclonal antibodies for a more reliable serological detection and identification of bacteria, as was demonstrated for LPS antigens of Erwinia carotovora subsp. atroseptlca (3).


Conclusions For viruses, over three million tests are done each year to index various types of plant propagative materials with ELISA in the Netherlands. Polyclonal antisera gave good results for all important viruses involved in the testing. The main problem in the development of a test was with optimizing the preparation of the plant sample extracts. In the future, attention will be given to improving the handling of large series by automated procedures. Compared to viruses, many problems are s t i l l associated with the routine indexing of plant materials for pathogenic bacteria. ELISA is less sensitive than immunofluorescence microscopy and both methods involve a risk of false positive reactions due to cross-reacting saprophytic micro-organisms, particularly when antisera are not properly tested. New methods are presented based on combining immunotrapping, isolation, and Identification of colonies directly in agar media. Of these methods, immuno-isolation combined with immunofluorescence colony staining especially has good prospects for detecting low concentrations of the pathogen and for efficient isolation of cross-reacting organisms. The availability of a representative collection of crossreacting organisms for a certain type of sample material is a major requirement in the research for typical antigens of the target bacterium, in order to produce more specific monoclonal or polyclonal antisera. Literature Cited 1. Barzic, M.R.; Trigalet, A. Agronomie 1982, 2, 389-398. 2. Beijersbergen, J.C.M.; Van der Hulst, C.Th.C. Neth. J . Pl. Path. 1982, 86, 277-283. 3. DeBoer, S.H.; McNaughton, M.E. Phytopathology 1987, 77, 828-832. 4. Bos, L. In Plant Health and Quarantine in International Transfer of Genetic Resources; Hewitt, W.B.; Chiarappa, L., Eds.; CRC Press Inc.: Cleveland, Ohio, 1977; pp. 39-69. 5. Clark, M.F. Ann. Rev. of Phytopathol. 1981, 19, 83-106. 6. Clark, M.F.; Adams, A.N. J . gen. Virol. 1977, 34, 475-483. 7. Delecolle, H.; Lot, H.; Michel, M.J. Phytoparasitica 1985, 13, 266-267. 8. Endel, E.A.; Van Vuurde, J.W.L. In EAPR Abstracts of Conference Papers and Posters; R. Rasmussen: Denmark,1987;pp.190-191. 9. Gugerli, P.; Gehriger, W. Potato Research 1980, 23, 353-359. 10. International Seed Testing Assiociation. Seed Sc. and Techn. 1985, 13, 299-355. 11. Janse, J.D.; Van Vaerenbergh, J . EPPO Bulletin 1987, 17, 1-10. 12. Koenig, R. J . gen. Virol. 1978, 40, 309-318. 13. Koenig, R. J . gen. Virol. 1981, 55, 53-62.

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14. Malin, M.E.; Roth, D.A.; Belden, E.L. Plant Disease 1983, 67, 645-647. 15. Marrou, J.; Messiaen,C.M.Proceedings of the International Seed Testing Association 1967, 32, 49-57. 16. Miller, H.J. Potato Research 1984, 27, 33-42. 17. Rohloff, I. Proceedings of the International Seed Testing Association 1967, 32, 59-63. 18. Schaad, N.W. Plant Disease 1982, 66, 885-890. 19. Schaad,N.W.;Donaldson, R.C. Seed S c i . and Technol. 1980, 8, 383-391. Biotechnology for Crop Protection Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.

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30,31. 21. Tijssen, P. Practice and Theory of Enzyme Immunoassays; Elsevier: Amsterdam 1985; pp. 541. 22. Trigalet, A.; Samson, R; Coleno, A. Proc. 4th Int. Conf. Plant Path. Bact. Angers, 1978, pp. 271-288. 23. Voller, A.; Bartlett, A.; Bidwell, D.E.; Clark, M.F.; Adams, A.N. J. gen. Virol. 1976, 33, 165-167. 24. Vruggink, H. Proceedings of the 4th International Conference on Plant Pathogenic Bacteria, Angers, 1978, pp. 307-310. 25. Vruggink, H.; Van Vuurde, J.W.L. In Methods in Phytobacteriology. Klement, Z . , Rudolph, K.; Sands,D.C.,Eds.; Akademiai Kiado: Budapest 1988. 26. Van Vuurde, J.W.L.; Van den Bovenkamp,G.W.;Birnbaum, Y. Seed Sci. & Technol. 1983, 11, 547-559. 27. Van Vuurde, J.W.L.; Van Henten, C. Seed Sci. and Technol. 1983, 11, 523-533. 28. Van Vuurde, J.W.L.; Maat, D.Z. Seed Sci. & Technol. 1983, 11, 505-513. 29. Van Vuurde, J.W.L.; Maat, D.Z. Neth. J. Pl. Path. 1985, 91, 3-13. 30. Van Vuurde, J.W.L.; Ruissen, M.A.; Vruggink, H. In Proc. 6th Int. Conf. on Plant Path. Bact., 1987, M. Nijhof, Dordrecht, pp. 835-842. 31. Van Vuurde, J.W.L. In Proc. 6th Int. Conf. on Plant Path. Bact., 1987, M. Nijhof, Dordrecht, pp. 799-808. 32. Van Vuurde, J.W.L. EPPO Bulletin 1987, 17, 139-148. RECEIVED

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