Edifenphos Resistance in Pyricularia oryzae and Drechslera oryzae

Chapter 17

Edifenphos Resistance in Pyricularia oryzae and Drechslera oryzae In Vitro Techniques for Detection and Biochemical Studies

Downloaded by CORNELL UNIV on October 17, 2016 | http://pubs.acs.org Publication Date: February 23, 1990 | doi: 10.1021/bk-1990-0421.ch017

D. Lalithakumari and P. Annamalai Centre for Advanced Studies in Botany, University of Madras, Guindy, Madras 600 025, India

Resistance to edifenphos (Hinosan, EDDP) in Pyricularia oryzae and Drechslera oryzae has been noticed in the field. EDDP resistant strains of P. oryzae and D. oryzae were obtained in vitro, by UV irradiation, adaptation and chemical mutagenesis. The EDDP resistant mutants were found to be stable and pathogenic. Morphological variations were not observed in EDDP mutants of P. oryzae while the field mutants of D. oryzae distinctly differed in morphology and pigmentation. The Mechanism of resistance to EDDP was presumed to be alteration in the membrane integrity and not in its site of action, as phosphatidylcholine remained unaffected in mutants. Plasmid mediated resistance to EDDP was presumed, as the intensity of plasmid bands was high in resistant mutants of P. oryzae and D. oryzae. P. oryzae mutants showed high sensitivity to Ziram and D. oryzae mutants to Mancozeb in crossresistance studies. The National Agricultural Research System in India brings out a number of new crop varieties and hybrids but it is well known that in sub-tropical and tropical climates crop resistance to obligate parasites does not last for more than 4 or 5 years. Modern plant breeding technology and improved crop improvement practices do not solve this problem. Hence chemical control is indispensable and highly effective in controlling many major diseases. Systemic fungicides have improved tremendously. There are many lines of evidence suggesting that systemic fungicides not only control the pathogen but also alter the host physiology thereby enhancing the defence mechanism of the host plant (1-3). Problems of pathogen resistance to conventional fungicides have been reported by many workers in India (4-7). But increasing reports of fungal resistance to systemic fungicides are left unnoticed or only very 0097-6156/90/0421-0249$06.00/0 © 1990 American Chemical Society

Green et al.; Managing Resistance to Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

250

MANAGING

RESISTANCE TO AGROCHEMICALS

few o c c u r r e n c e s a r e r e p o r t e d so f a r (7,8-12). Work on molecular b a s i s of r e s i s t a n c e and c o n t r o l measures i s c o m p l e t e l y l a c k i n g i n India. Hence, the p r e s e n t i n v e s t i g a t i o n emphasizes t h e mechanism, molecular b a s i s o f f u n g i c i d e r e s i s t a n c e and an easy method t o c o n t r o l or prevent f u n g i c i d e r e s i s t a n c e i n P y r i c u l a r i a oryzae and D r e c h s l e r a o r y z a e a g a i n s t e d i f e n p h o s (EDDP, H i n o s a n ) . MATERIALS

AND METHODS


Fungal strains: Pyricularia oryzae and D r e c h s l e r a oryzae (parent), sensitive to edifenphos were m a i n t a i n e d on potato dextrose agar (PDA). The e d i f e n p h o s - r e s i s t a n t mutants were maintained on edifenphos-amended PDA. For the present study, uniformaly 8 and 10-day-old c u l t u r e s o f D_. o r y z a e and oryzae, r e s p e c t i v e l y , were used t h r o u g h o u t the s t u d y . Mutagenesis: C h e m i c a l mutagens e t h y l methane s u l p h o n a t e (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (NTG) were used as mutagenic agents. Ungerminated c o n i d i a o f £ . o r y z a e and D_. o r y z a e a t 2.5 x 10 c o n i d i a / m l were t r e a t e d w i t h EMS, 6 mg/ml i n pH 7.0 sodium phosphate buffer and NTG, 0.5mg/ml i n pH 9.0 Tris maleic acid buffer ( 1 3 ) . T r e a t e d c o n i d i a were p l a t e d on EDDP-amended medium c o n t a i n i n g EDDP 5 t i m e s t h e e f f e c t i v e dose r e q u i r e d t o i n h i b i t 50% of p a r e n t s t r a i n (ED,.^). C o l o n i e s appeared on the amended medium were p i c k e d out and m a i n t a i n e d on fungicide-amended medium. 5

The Ultraviolet light induced mutants were obtained by e x p o s i n g the c o n i d i a l s u s p e n s i o n o f ]?. o r y z a e and D_. o r y z a e t o UV light a t wavelength o f 254 nm f o r 30 m i n u t e s . The UV-exposed conidia were a g a i n p l a t e d on EDDP-amended medium as mentioned above and s u r v i v i n g mutants were i s o l a t e d . Adapted mutants: Adapted mutants were s e l e c t e d by the n a t u r a l s c r e e n i n g o f 0.5 x 10 /ml c o n i d i a o f P_. o r y z a e and D_. o r y z a e over the EDDP-amended medium. C o l o n i e s o b t a i n e d thus on f i v e times higher c o n c e n t r a t i o n o f the E D Q dose o f s e n s i t i v e strain were f u r t h e r t r a i n e d t o grow on h i g h e r c o n c e n t r a t i o n s . 5

5

Field mutants: Four E D D P - r e s i s t a n t mutants of £ . oryzae were obtained from EDDP-treated paddy r i c e f i e l d s showing no disease control. Test f o r s t a b i l i t y and p a t h o g e n i c i t y : A l l o f the i s o l a t e d EDDPresistant mutants o f both P_. o r y z a e were grown on fungicide-free and amended medium f o r 10 t r a n s f e r s , t o t e s t t h e i r stability of resistance. Additionally, p a t h o g e n i c i t y o f a l l mutants was confirmed on the r i c e c u l t i v a r IR 50. O n l y s t a b l e and pathogenic mutants were chosen f o r f u r t h e r s t u d i e s . S t a b l e and v i r u l e n t mutants: F o r £ . o r y z a e , one EMS (POLR-1), t h e adapted mutants (POLR-2 and POLR-3) and one UV mutant (POLR-4) and for £. o r y z a e , one EMS mutant (DOLR-1) and f o u r field mutants (DOFR-1, DOFR-2, DOFR-3 and DOFR-4) were chosen based on their s t a b i l i t y and p a t h o g e n i c i t y .


17.

LALITHAKUMARI AND ANNAMALAI

Edifenphos Resistance

Morphological Characterization s e n s i t i v e (parent) s t r a i n s

of

EDDP-resistant

mutants

All of t h e t e s t e d m u t a n t s were grown on fungicide-free (PDA) and compared w i t h s e n s i t i v e s t r a i n f o r g r o w t h r a t e , morphology, p i g m e n t a t i o n and c o n i d i a l p r o d u c t i o n . Biochemical Characterization s e n s i t i v e (parent) s t r a i n s

of

EDDP-resistant

251 and

medium colony

mutants

and

Rate of e f f l u x of e l e c t r o l y t e s : E f f l u x of e l e c t r o l y t e s from the fungal mycelium was m e a s u r e d u s i n g a T y p e CM82 T c o n d u c t i v i t y bridge with a dip electrolyte c e l l (1). The conductance was expressed a s s p e c i f i c c o n d u c t a n c e i n u m h o s / c m / n / g / d r y wt o f the mycelium.


2

Estimation o f t o t a l DNA, RNA a n d p r o t e i n : I s o l a t i o n of degraded DNA f r a c t i o n was c a r r i e d o u t by t h e m o d i f i e d m e t h o d o f M u n r o and Fleck(14). DNA was e s t i m a t e d by G i l e s a n d Myers(15) further m o d i f i e d by L a l i t h a k u m a r i e t a l . ( 1 6 ) . T o t a l RNA was e s t i m a t e d by the method of Wu(17). T o t a l p r o t e i n was e s t i m a t e d by t h e method of Lowry e t a l . ( 1 8 ) . E x t r a c t i o n o f u n d e g r a d e d DNA: The e x t r a c t i o n o f u n d e g r a d e d DNA was carried out by using Marmur's m e t h o d ( 1 9 ) . The DNA strands recovered at t h e e n d w e r e d i s s o l v e d i n 10 m l of sodium saline citrate ( S S C ; N a C l , 0.15M; t r i s o d i u m c i t r a t e , 0.15 M a t pH 7 . 0 ) . Aliquots of undegraded DNA i n SSC s o l u t i o n w e r e taken for UV s p e c t r a l a n a l y s i s t h a t w e r e c a r r i e d o u t b e t w e e n 220 a n d 320 nm a n d compared w i t h t h e s p e c t r u m o b t a i n e d w i t h c a l f thymus. Determination o f t h e DNA b a s e c o m p o s i t i o n : Two d i f f e r e n t methods w e r e u s e d f o r t h e e s t i m a t i o n o f DNA b a s e c o m p o s i t i o n , G u a n i n e p l u s C y t o s i n e (GC) c o n t e n t . (a) (b)

UV a b s o r p t i o n r a t i o OD 260 / OD 280(20.). Thermal d e n a t u r a t i o n method(21).

Electrophoretic p a t t e r n of p r o t e i n s : P r o t e i n p a t t e r n of mutants and parent strains was analysed by polyacrylamide gel e l e c t r o p h o r e s i s (PAGE) u s i n g t h e m e t h o d o f D a v i s ( 2 2 ) . Estimation of t o t a l l i p i d s and p h o s p h o l i p i d s : The total lipid content was e s t i m a t e d a c c o r d i n g t o T s u d a e t a l . ( 2 3 ) a n d purified a c c o r d i n g t o B l i g h and D y e r ( 2 4 ) . P h o s p h o l i p i d s were separated by thin l a y e r c h r o m a t o g r a p h y (TLC) m e t h o d ( 2 5 ) . The phospholipids w e r e q u a n t i f i e d by e s t i m a t i n g t h e p h o s p h o r o u s c o n t e n t ( 2 6 ) . Isolation

of plasmid

f r o m P.

o r y z a e a n d D.

oryzae p r o t o p l a s t s

Isolation of p r o t o p l a s t s : Pure suspensions of p r o t o p l a s t s from P_. o r y z a e a n d D_. o r y z a e w e r e o b t a i n e d u s i n g t h e m e t h o d o f Hashiba and Yamada(27). Mycelia from shaken c u l t u r e s were harvested aseptically by vacuum f i l t r a t i o n and washed t w i c e w i t h distilled water and o n c e w i t h 0.6 M S u c r o s e m a n n i t o l osmotic stabilizer.


252

MANAGING RESISTANCE TO AGROCHEMICALS


One gram m y c e l i a l mat was weighed and suspended i n a 10 ml enzyme mixture (containing c e l l u l a s e 30mg/ml; p e c t i n a s e lOmg/ml; 3glucuronidase O.lml/ml and c h i t i n a s e 25mg /ml i n 0.6 M Sucrose m a n n i t o l pH 5.5) i n a 100ml Erlenmeyer f l a s k . The f l a s k was p l a c e d on a r e c i p r o c a l shaker a t 75 s t r o c k s / m i n . a t 28°C f o r 3 h. The c u l t u r e was f i l t e r e d through s i x l a y e r s of cheese c l o t h to remove mycelial fragments and the f i l t r a t e was c e n t r i f u g e d a t 1,000 rpm for 5 min t o remove t r a c e s of the enzymes. Intact protoplasts were f u r t h e r s e p a r a t e d from m y c e l i a l fragments and c e l l d e b r i s by an aqueous two-phase system. P r o t o p l a s t s were counted using a haemocytometer and e x p r e s s e d as number of p r o t o p l a s t s per gram dry or fresh weight. Isolated protoplasts were checked for r e g e n e r a t i o n on Czapek's (Dox) y e a s t e x t r a c t agar medium. Isolation of p l a s m i d DNA from the p r o t o p l a s t s of P. oryzae and D. o r y z a e : For the i s o l a t i o n of p l a s m i d DNA i n P_. oryzae, the method of T a k a i e t a l . ( 2 8 ) was f o l l o w e d and f o r D_. o r y z a e Kistler and Leong's(29) method was followed. Agarose gel e l e c t r o p h o r e s i s : From the purified protoplasts of P_. o r y z a e and D_. o r y z a e p l a s m i d DNA were i s o l a t e d by preparative 0.7% agarose gel electrophoresis at 60 V for 4 h. The electrophoresis b u f f e r c o n t a i n e d 0.8M T r i s , 0.4M sodium acetate, 0.04M EDTA and a c e t i c a c i d 1ml (pH 8.3). Sample was mixed with 1/10 volume of 25% F i c o l l (Sigma) and 0.25% bromo p h e n o l b l u e dye was added before electrophoresis. Staining of the gel was p e r f o r m e d u s i n g e t h i d i u m bromide, 5Ug/ml. p l a s m i d DNA was viewed and photographed u s i n g RP4 p o l a r o i d camera. C u r i n g of p l a s m i d was done u s i n g e t h i d i u m b r o m i d e ( 3 0 ) . Counter measure f o r e d i f e n p h o s

resistance

C r o s s r e s i s t a n c e s t u d i e s : A l l of the e d i f e n p h o s - r e s i s t a n t s t r a i n s were t e s t e d f o r t h e i r c r o s s - r e s i s t a n c e t o r e l a t e d and unrelated systemic and c o n t a c t f u n g i c i d e s . The growth r a t e of both mutant and s e n s i t i v e s t r a i n s were compared and t h e i r ED values were estimated. The r e s i s t a n c e l e v e l was e x p r e s s e d as Q v a l u e (ratio of E D of s e n s i t i v e s t r a i n / E D Q of mutant s t r a i n s ) ( 3 1 ) . 5 Q

5

RESULTS Morphology, Growth and S p o r u l a t i o n : T h e r e were no d i f f e r e n c e s in color and c o l o n y morphology of e d i f e n p h o s - r e s i s t a n t mutants and sensitive strain of ]?. o r y z a e . However e d i f e n p h o s - r e s i s t a n t mutants showed a slow growth r a t e when compared to sensitive strain of J?, o r y z a e . In D_. o r y z a e , the f i e l d mutants (DOFR-1, DOFR-2, DOFR-3 and DOFR-4) d i f f e r e d i n morphology and mycelial growth, showed p u f f y and l o b b e d growth ( F i g . 1 and 2) on the fungicide f r e e medium and c o l o r changed from g r e e n i s h black to blackish brown. Sporulation was reduced i n both I?, o r y z a e and D_. o r y z a e mutants than s e n s i t i v e s t r a i n . A l l the t e s t e d oryzae and D_. o r y z a e mutants were as i n f e c t i v e as the s e n s i t i v e s t r a i n s of t h e s e pathogens.



17. LALITHAKUMARI AND ANNAMALAI


Figure 1. Colony morphology of sensitive strain mutants of D. oryzae.

Figure 2. Colony morphology of field resistant mutants of D. oryzae.


253

254

MANAGING RESISTANCE TO AGROCHEMICALS Biochemical Characterization (parent) s t r a i n s

of r e s i s t a n t mutants

and

sensitive

Rate o f e f f l u x o f e l e c t r o l y t e s : The r e s u l t s i n T a b l e I clearly indicates a s i g n i f i c a n t reduction i n e f f l u x of e l e c t r o l y t e s from all t h e edifenphos r e s i s t a n t mutants of £. oryzae and £. oryzae throughout the observation period. Table I . Rate

of e f f l u x of e l e c t r o l y t e s of s e n s i t i v e strain r e s i s t a n t mutants of o r y z a e a n d D_. o r y z a e


Specific

2 P oryzae Parent(Sensitive) POLR-1 POLR-2 POLR-4 D. o r y z a e Parent(Sensitive) DOLR-1 DOFR-1 DOFR-2

2

c o n d u c t a n c e umhos/cm / n / g / d r y of mycelium I n c u b a t i o n Time (hrs) 8 6 4

and

wt

12

2750 1460 1450 860

4800 2220 2210 1090

5206 3403 3250 1318

6173 3611 3500 1355

8039 5277 5200 1864

2454 2370 2352 2100

3818 2727 2647 2450

4363 3272 3529 2860

4818 4045 4117 3215

5636 5063 4803 3800

Macromolecular synthesis:The macromolecular contents increased i n a l l of the mutants. T h e T o t a l DNA c o n t e n t ( T a b l e I I ) was o b s e r v e d to be h i g h e r i n t h e e d i f e n p h o s r e s i s t a n t m u t a n t s . UV-induced oryzae mutants s h o w e d o n l y 2 0 % i n c r e a s e o f DNA over sensitive strain. S i m i l a r l y D o r y z a e m u t a n t s a l s o showed an i n c r e a s e i n t h e DNA c o n t e n t . Among t h e p h o s p h o l i p i d s t h e i n c r e a s e i n p h o s p h a t i d y l c h o l i n e was s i g n i f i c a n t i n t h e r e s i s t a n t m u t a n t s . Table

I I . Macromolecular content r e s i s t a n t mutants of

of sensitive strain o r y z a e a n d D_. o r y z a e dry

weight Phospholipids

P. o r y z a e Parent (sensitive) POLR-1 POLR-2 POLR-3 D. o r y z a e Parent (sensitive) DOLR-1 DOFR-1 DOFR-2

DNA mg/g

RNA mg/g

Protein mg/g

Lipid %

PEug

pcyg

3.19 4.92 4.82 3.82

2.50 3.25 3.57 3.20

11.80 13.60 15.72 15.65

15.10 14.00 14.60 14.90

0.720 0.830 0.900 0.890

0.840 0.930 0.950 0.930

7.80 9.75 10.00 9.70

4.60 5.70 5.75 5.85

15.30 20.10 21.10 20.10

10.20 11.00 13.00 12.00

0.850 0.950 0.975 0.965


1.220 1.350 1.385 1.360

and


255


Electrophoretic protein pattern: The e l e c t r o p h o r e t i c pattern of p r o t e i n ( T a b l e I I I ) r e v e a l e d e x t r a bands i n two o f t h e e d i f e n p h o s resistant m u t a n t s (POLR-1 a n d P O L R - 4 ) o f ]P. o r y z a e . POLR-2 d i d n o t show a n y e x t r a b a n d . I n p_. o r y z a e t h e r e was n o d i f f e r e n c e i n t h e number o f p r o t e i n b a n d s b u t t h e i n t e n s i t y was g r e a t e r i n a l l the e d i f e n p h o s - r e s i s t a n t mutants. T a b l e I I I . E l e c t r o p h o r e t i c p r o t e i n p a t t e r n o f s e n s i t i v e s t r a i n and r e s i s t a n t m u t a n t s o f P_. o r y z a e a n d D_. o r y z a e


T o t a l No. o f b a n d s P. o r y z a e Parent (Sensitive) POLR-1 POLR-2 POLR-4 D. o r y z a e Parent (Sensitive) DOLR-1 DOFR-1 DOFR-2 Characterization (Tin)

observed

10 13 10 13 9 9 9 9

o f u n d e g r a d e d DNA f o r GC% a n d m e l t i n g

temperature

The r e s u l t s i n T a b l e I V show a v a l u e o f 50.2 a n d 4 9 . 2 a s GC% f o r the parent strain o f j ? . o r y z a e b y UV a b s o r p t i o n and thermal d e n a t u r a t i o n methods r e s p e c t i v e l y . S i m i l a r l y , i n D_. o r y z a e p a r e n t strain t h e GC% was 57.2 a n d 58.2 b y b o t h m e t h o d s . T h e GC% was higher i n t h e r e s i s t a n t mutant than i n t h e p a r e n t s t r a i n o f both P. o r y z a e a n d p_. o r y z a e . T h e m e l t i n g t e m p e r a t u r e (Tm) was 8 7 . 0 , 90.0, 91.0 a n d 96.0°C r e s p e c t i v e l y f o r £ . o r y z a e p a r e n t , P O L R - 1 , POLR-2 a n d POLR-4. F o r D_. o r y z a e i t was 8 7 , 8 8 , 9 0 , 90°C r e s p e c t i v e l y f o r p a r e n t , DOLR-1, DOFR-1, DOFR-2. Table

I V . C h a r a c t e r i z a t i o n o f u n d e g r a d e d DNA f o r GC% a n d m e l t i n g t e m p e r a t u r e (Tm) o f P_. o r y z a e a n d D_. o r y z a e GC% o f u n d e g r a d e d UV

P. o r y z a e Parent (Sensitive) POLR-1 POLR-2 POLR-4 D. o r y z a e Parent (Sensitive) DOLR-1 DOFR-1 DOFR-2

absorbancy method

Thermal denaturation method

DNA Tm

value (°C)

50.2 51.4 55.1 65.4

49.2 52.0 52.9 65.1

87 90 91 96

57.2 61.4 61.8 60.8

58.2 61.8 61.2 59.8

87 88 90 90


256

MANAGING RESISTANCE TO

AGROCHEMICALS

Isolation of P l a s m i d DNA from the p r o t o p l a s t s of P. oryzae and D. o r y z a e : Recovery of p r o t o p l a s t s on an average was 1.6 x 1 0 in P_. o r y z a e and 2 x 1 0 i n p_. o r y z a e ( F i g . 3 ). The p r o t o p l a s t s of P_. o r y z a e were d i s t i n c t l y c i r c u l a r and I), o r y z a e p r o t o p l a s t s were b i g g e r than £ . o r y z a e p r o t o p l a s t s . s

5

A n a l y s i s of e d i f e n p h o s - r e s i s t a n t mutant and s e n s i t i v e strains of o r y z a e and D_. o r y z a e ( F i g . 4 , 5a andSb)showed a s i n g l e p l a s m i d DNA band i n the agarose gel electrophoresis. F u r t h e r , the intensity of P l a s m i d bands was more i n both .P. o r y z a e and D_. o r y z a e r e s i s t a n t mutants than the s e n s i t i v e s t r a i n . Counter

measure f o r e d i f e n p h o s


Cross r e s i s t a n c e

resistance

studies:

The results i n T a b l e V show t h a t the c h e m i c a l mutant of J?, o r y z a e (POLR-1) was h i g h l y s e n s i t i v e t o Carbendazim, Ziram and Mercuric chloride. Adapted mutant POLR-2 was found s e n s i t i v e to Bitertanol, Ziram, and Mancozeb. The UV mutant POLR-4 was s e n s i t i v e t o W e t t a b l e s u l p h u r , Ziram and Mancozeb. EMS mutant of p_. o r y z a e (DOLR-1) showed high sensitivity t o Mancozeb and Bitertanol while the two r e s i s t a n t f i e l d strains (DOFR-1 and DOFR-2) were s e n s i t i v e o n l y t o Mancozeb. The r e s i s t a n c e level (Q v a l u e ) was l e s s than one f o r the above c h e m i c a l s . Table

V.

Cross r e s i s t a n c e to f u n g i c i d e s Q value

Fungicide

.p. o r y z a e

Benomyl Carbendazim Bitertanol IBP Pyroquilon Thiophanate methyl Mancozeb Ziram Copper oxy chloride Wettable sulphur Methoxy e t h y l mercury chloride Mercuric chloride * Negative

POLR-1

POLR-2

1.5 0.6* 3.2 2.1

1.8 2.8 0.7* 2.0

POLR-3 2.0 1.7 1.6 3.0

D_. oryzae POLR-4 1.5 1.3 1.5 1.8

-

DOLR-1 DOFR-1 DOFR-2 1.20 1.15 0.50* 1.39 1.16

1.11 1.05 1.60 1.13 1.04

1.21 1.03 4.00 1.21 1.15

-

-

-

2.4 3.8 0.5*

2.1 0.8* 0.8*

1.2 1.0* 0.9*

1.1 0.8* 0.5*

1.09 0.80* 1.38

1.26 0.80* 2.69

1.07 0.90* 1.76

1.5

1.2

1.5

1.5

1.23

1.50

1.19

1.0

1.0

0.8*

0.3*

1.40

1.44

1.23

3.5

1.0

1.0

1.2

1.5

1.7

0.9*

-

-

0.8*

-

-

correlation



17.

LALITHAKUMARI AND ANNAMA1AI

Figure

3.

Protoplasts


of D.oryzae

(320

x).


257

258



LANES

1

2

3

4

5

6

7

8

Figure 4. Agarose gel electrophoresis of Plasmid D N A of P. oryzae.

LANES

1

2

LANES

12

3

4

Figure 5. Agarose gel electrophoresis of Plasmid D N A of D. oryzae.




259

DISCUSSION Edifenphos (EDDP) i s e f f e c t i v e l y used f o r the c o n t r o l o f both yzae and D_. o r y z a e i n I n d i a . L e v e l of r e s i s t a n c e found i n the field o f EDDP i s u s u a l l y r a t h e r low and t h e r e f o r e i t i s difficult t o d e t e c t and measure t h e EDDP- r e s i s t a n c e . Since fungicidal a c t i v i t y and f u n g a l r e s i s t a n c e a r e e a s i e r t o e v a l u a t e in v i t r o , t h e p r e s e n t i n v e s t i g a t i o n employed in_ v i t r o methods t o determine t h e p r o b a b i l i t y l e v e l , mechanism and m o l e c u l a r b a s i s of resistance and c o n t r o l measures t o break r e s i s t a n c e i n £ . o r y z a e and D_. o r y z a e t o EDDP. o r


5

Screening o f 0.5 x 10 /ml c o n i d i a of P_. o r y z a e and D_. o r y z a e on EDDP-amended media r e v e a l e d t h e v a r i a b i l i t y in sensitivity among t h e c o n i d i a , b e s i d e s the development o f two c o l o n i e s o f P. oryzae r e s i s t a n t t o EDDP. S e l e c t i o n o f mutants r e s i s t a n t t o the Kasugamycin, K i t a z i n and EDDP from a l a r g e number o f c o n i d i a has been r e p o r t e d by K a t a g i r i and U e s u g i ( 3 2 ) . These two c o l o n i e s (POLR-2 and POLR-3) were f u r t h e r t r a i n e d t o grow i n higher concentration o f EDDP by an a d a p t a t i o n t e c h n i q u e . Many colonies of D_. o r y z a e d e v e l o p e d on h i g h c o n c e n t r a t i o n o f EDDP but they d i d not grow w e l l i n the absence of f u n g i c i d e . No r e s i s t a n t mutants of D_. o r y z a e developed by t h e UV irradiation technique. Georgopoulos et_ a_l. (33) a l s o were unable to obtain resistance mutants o f U s t i l a g o maydis by UV i r r a d i a t i o n . S e l e c t e d _in v i t r o mutants o f P_. o r y z a e and p_. o r y z a e i n d u c e d by EMS treatment, UV irridation and a d a p t a t i o n , s e r v e d as good examples o f EDDPr e s i s t a n c e owing t o t h e i r s u r v i v a l i n t h e absence o f f u n g i c i d e and their a b i l i t y t o i n f e c t on paddy r i c e . E D D P - r e s i s t a n t mutants o f P_. o r y z a e d i d n o t show any v a r i a t i o n i n c o l o n y morphology and c o l o r , but t h e f i e l d r e s i s t a n t mutants o f D_. o r y z a e showed a p u f f y and lobbed growth w i t h changes i n p i g m e n t a t i o n . A l l o f t h e resistant mutants o f P_. o r y z a e and D_. o r y z a e showed slight reduction i n sporulation. The r e d u c t i o n i n s p o r u l a t i o n by resistant mutants has been r e p o r t e d by many workers(34-37). Pathogenicity o f a l l t h e r e s i s t a n t mutants o f JP. o r y z a e and D_. o r y z a e t o r i c e p l a n t s proved t h e i r f i t n e s s i n t h e f i e l d t o compete w i t h t h e p a r e n t s t r a i n i n t h e absence as w e l l as i n t h e presence o f EDDP. Hence, t h e s e l e c t e d mutants i n the present i n v e s t i g a t i o n s e r v e d as v e r y good examples of EDDP r e s i s t a n c e . V a r i o u s c e l l u l a r c o n t e n t s analysed,showed s i g n i f i c a n t i n c r e a s e in a l l the m a c r o m o l e c u l a r c o n t e n t s v i z . DNA, RNA P r o t e i n , lipids and p h o s p h o l i p i d s of the E D D P - r e s i s t a n t mutants o f o r y z a e and p_. o r y z a e . The e f f l u x of e l e c t r o l y t e s was reduced i n the resistant mutants when compared t o c o n t r o l . In i n t e r p r e t i n g t h e r e s u l t s , i t s h o u l d be r e a l i z e d t h a t t h e p h o s p h o l i p i d s , t h e t a r g e t site o f i n h i b i t i o n f o r e d i f e n p h o s seem t o be u n a f f e c t e d i n t h e mutants as e v i d e n c e d from t h e h i g h e r v a l u e s o f p h o s p h a t i d y l c h o l i n e compared s e n s i t i v e s t r a i n . These d a t a i n d i c a t e t h a t t h e mechanism of r e s i s t a n c e t o edifenphos i s not r e l a t e d t o the t a r g e t s i t e of e d i f e n p h o s . De waard and Van N i s t e l r o o y ( 3 8 ) a l s o r e p o r t e d t h a t t h e mechanism o f r e s i s t a n c e i n ]?. o r y z a e t o pyrazophos (PP) was n o t related t o t h e change of t a r g e t s i t e o f PP. I t seems p r o b a b l e that resistance i n .P. o r y z a e and D_. o r y z a e to edifenphos


260



influences cell membrane p e r m e a b i l i t y a s evidenced from the reduction i n the efflux of electrolytes from the resistant mutants. S i m i l a r l y , R a n k et_ a_l.( 39) d e m o n s t r a t e d that decreased membrane p e r m e a b i l i t y was the mechanism of resistance in Saccharomycos cerevisiae resistant to various toxicants, in A s p e r g i l l u s n i d u l a n s r e s i s t a n t t o f e n a r i m o l (40) and d i c a r b o x i m i d e a n d b e n z i m i d a z o l e r e s i s t a n t m u t a n t s o f G e r l a c h i a n i v a l i s ( 4 1 ) . The a c q u i r e d m e c h a n i s m o f r e s i s t a n c e i n D_. o r y z a e by i n _ v i t r o mutants is identical w i t h the n a t u r a l mechanism of r e s i s t a n c e i n field r e s i s t a n c e m u t a n t s o f D_. o r y z a e i s o l a t e d f r o m t h e f i e l d . There are many r e p o r t s on the chromosomal control of resistance to a g r i c u l t u r a l f u n g i c i d e s (42-44). Chromosomal gene i n v o l v e m e n t i n f u n g i c i d e r e s i s t a n t has been r e p o r t e d so f a r owing to the reason t h a t t o x i c a n t s mostly a c t o u t s i d e the mitochondria. In eucaryotic cells, m i t o c h o n d r i a l DNA c a n n o t be involved in resistance of the toxicants acting outside the mitochondria. Involvement of p l a s m i d s , the extrachromosomal genes i n r e s i s t a n c e in common in bacteria (45 48). But, plasmids have been recognised q u i t e r e c e n t l y i n f u n g i , involvement of plasmid in benzimidazole resistance i n Mycospherella musicaola (49) and Neurospora c r a s s a (50) have a l r e a d y been r e p o r t e d . In addition, Saccharomyces c e r e v i s i a e r e s i s t a n t t o o l i g o m y c i n was r e p o r t e d to be d u e t o e x t r a c h r o m o s o m a l DNA ( 5 1 ) . B e s i d e s , a linear plasmids h a v e b e e n d e s c r i b e d i n a number o f F u s a r i u m s p . ( 5 2 ) , C o c h l i o b o l u s heterostrophus (53) and A s c o b o l u s immersus ( 5 4 ) , however, no definite r o l e h a s b e e n e x p l a i n e d . P l a s m i d DNA was isolated from s e n s i t i v e s t r a i n a n d r e s i s t a n t m u t a n t s o f P_. o r y z a e a n d D_. o r y z a e , but i n the r e s i s t a n t m u t a n t s o f P_. o r y z a e and £. oryzae the intensity o f p l a s m i d DNA was m o r e t h a n t h e s e n s i t i v e s t r a i n . The high i n t e n s i t y o f p l a s m i d DNA b a n d s i n t h e r e s i s t a n t m u t a n t s may be due t o i n c r e a s e d number o f p l a s m i d c o p i e s which might have resulted from r a p i d a m p l i f i c a t i o n of p l a s m i d s i n the presence of edifenphos. T h i s h i g h i n t e n s i t y i n p l a s m i d s was a l s o o b s e r v e d in p l a n t o m y c i n - r e s i s t a n t mutants of Xanthomonas c a m p e s t r i s pv. o r y z a e (55). Similar increase i n copy number of plasmids due to resistance h a s b e e n r e p o r t e d by C l e w e l l ( 4 8 ) a n d N o r d s t r o m (56). Further, Annamalai (57) has r e p o r t e d t h a t c u r i n g w i t h ethidium b r o m i d e f o r 48 h i n d a r k n e s s r e s u l t e d i n t h e l o s s o f r e s i s t a n c e t o edifenphos i n D_. o r y z a e . P r o t o p l a s t s i s o l a t e d f r o m p l a s m i d cured p r o t o p l a s t s d i d n o t show p l a s m i d DNA b a n d s u n d o u b t e d l y confirming the d e f i n i t e i n v o l v e m e n t o f p l a s m i d DNA i n e d i f e n p h o s resistance in P_. o r y z a e a n d p_. o r y z a e . T h e r e a r e s e v e r a l r e p o r t s on plasmid mediated resistance to various toxicants. For instance, Escherichia coli r e s i s t a n t t o K a s u g a m y c i n by Yoshikawa et a l . (58), Xanthomonas campestris pv. vesicatoria resistant to Kanamycin and Neomycin ( 5 9 ) , Pseudomonas a e r u g i n o s a r e s i s t a n c e t o chloramphenicol (60), Flexibacter spp. and Agrobacterium tumifaciens resistant to tetracycline (61). Although the specificity o f p l a s m i d s and m o l e c u l a r w e i g h t of p l a s m i d s in the p r e s e n t i n v e s t i g a t i o n c o u l d n o t be e x p l a i n e d , i t i s c o n f i r m e d t h a t plasmid coded r e s i s t a n c e can occur i n the non-site specific resistance m e c h a n i s m o r i n membrane p e r m e a b i l i t y o f f u n g i . Cross r e s i s t a n c e s t u d i e s were c a r r i e d out t o s e l e c t a s u i t a b l e chemical for b r e a k i n g t h e E D D P - r e s i s t a n c e . The r e s u l t s on t h e sensitivity




261

of selected E D D P - r e s i s t a n t mutants o f P_. o r y z a e and D_. o r y z a e against unrelated fungicides were interesting. Also the pleiotropic effect observed regarding cross-resistance to unrelated compounds i n the present investigation confirm the mechanism of a c t i o n t o decreased membrane p e r m e a b i l i t y . The overall c o n c l u s i o n on t h e type o f m u t a t i o n i n P_. o r y z a e , and p_. o r y z a e t o e d i f e n p h o s was presumed t o be p l e i o t r o p i c mutation. Because, such a mutation may n o t o n l y cause resistance to unrelated compounds ( 6 2 ) . Such p l e i o t r o p i c mutation has been r e p o r t e d f o r r e s i s t a n c e t o t r i a r i m o l i n B o t r y t i s c i n e r e a (63) and imazalil i n Aspergillus n i d u l a n s ( 6 4 ) . A l l o f t h e mutants o f JL* y z a e were s e n s i t i v e t o Ziram and D^. o r y z a e t o Mancozeb though number of chemicals t o which each mutant showed varied sensitivity.


o r

Cross resistance i n an organism t o two d i f f e r e n t chemicals cannot be assumed w i t h o u t e v i d e n c e that sensitivity t o both chemicals i s controlled by t h e same gene ( 6 2 ) , b u t i t does influence the s e l e c t i o n of alternate chemicals f o r immediate control o f t h e pathogen i n t h e f i e l d . F o r a c o u n t r y like India, information on a l t e r n a t e c h e m i c a l s t o overcome d i s e a s e control failure i s very i m p o r t a n t as t h e f a r m e r s cannot afford yield l o s s e s a t any c o s t . The p r e s e n t o b s e r v a t i o n on c r o s s r e s i s t a n c e t o unrelated compounds have been confirmed by many workers (55, 65-70). I t i s , t h e r e f o r e , concluded that, t o overcome d i s e a s e c o n t r o l f a i l u r e o f £ . o r y z a e due t o E D D P - r e s i s t a n c e , Ziram may be used as an a l t e r n a t e chemical. I n D_. o r y z a e Mancozeb is recommended as an a l t e r n a t e chemical to break edifenphos resistance. The mechanism o f c r o s s r e s i s t a n c e i s not c l e a r l y u n d e r s t o o d from t h e s e r e s u l t s . ACKNOWLEDGMENTS We a r e e x t r e m e l y g r a t e f u l t o our D i r e c t o r , P r o f . A.Mahadevan, f o r h i s encouragement and t o t h e Department o f S c i e n c e and T e c h n o l o g y Govt, o f I n d i a f o r f u n d i n g t h e above work.

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Edifenphos Resistance in Pyricularia oryzae and Drechslera oryzae

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