Southeast Asian Fisheries Development Center Aquaculture Department
SEAFDEC/AQD Institutional Repository http://repository.seafdec.org.ph
Institutional Reports Quarterly Research Reports
1980
In-vitro effect of fungicides on hyphal
growth and sporogenesis of Lagenidium sp. isolated from Penaeus monodon
larvae and Scylla serrata eggs
Lio-Po, G. D.
Aquaculture Department, Southeast Asian Fisheries Development Center
Lio-Po, G. D., Sanvictores, M. E. G., Baticados, M. C. L., & Lavilla, C. R. (1980). In-vitro effect of fungicides on hyphal growth and sporogenesis of Lagenidium sp. isolated from Penaeus monodon larvae and Scylla serrata eggs. SEAFDEC Aquaculture Department Quarterly Research Report, 4(4), 1–6.
http://hdl.handle.net/10862/2377
Downloaded from http://repository.seafdec.org.ph, SEAFDEC/AQD's Institutional Repository
In -v itro effect o f fungicides on hyphal growth and sporogenesis o f Lagen idium sp. isolated from Penaeus m o n o d o n larvae and S cylla serrata eggs
G .D . L io -P o , M .E .G . S anvictores, M .C .L . Baticados and C .R . L avilla
T h e s e n s itiv ity o f L a g e n id iu m sp. is o la te d f r o m P en ae us m o n o d o n (is o la te F 1 6 -3 0 ) a n d S c y lla s e rra ta (is o la te F 1 1 1 -2 2 ) t o t h ir t y - f o u r a n tim y c o t ic c o m p o u n d s w a s d e te r m in e d . M y c o s t a t ic e ffe c ts w e re e v a lu a te d f r o m o b s e rv a tio n s o f c h e m ic a ls o n th e d e v e lo p m e n t o f ve sicle s, z o o spores and m y c e lia l g r o w th .
T h e rates o f m y c e lia l g r o w th a fte r 4 8 h r in c u b a tio n in f o u r d if f e r e n t m e d ia are p re s e n te d in T a b le 1. A n a ly s is o f va ria n c e o f th e g r o w th rates o f te s t fu n g i s h o w e d s ig n ific a n t d iffe re n c e s (P < 0 .0 1 ) . D u n c a n 's m u ltip le ran ge te s t ( P < 0 .0 5 ) s h o w e d t h a t n u t r ie n t agar is th e c o m m o n m e d iu m t h a t a llo w e d a c c e le ra te d g r o w th f o r b o t h iso la te s .
E x p e rim e n ts w ith o rg a n ic so lv e n ts used in s u b s e q u e n t tr ia ls reve a le d t h a t e th a n o l and d im e th y lfo r m a m id e possess in h e r e n t a n tim y c o t ic p ro p e rtie s (T a b le 2 ) . T h e ir use, th e r e fo r e , was lim ite d t o m a x im u m levels o f 5 ,0 0 0 m g /l f o r d im e t h y lfo r m a m id e a n d 5 0 0 m g /l f o r e th a n o l t o m in im iz e c o n t r ib u t o r y in h ib it in g e ffe c ts t h a t m ig h t m a sk th e r e la tiv e f u n g it o x ic a c t iv it y o f c o m p o u n d s b e in g assayed.
T h e s p e c ific a c tiv itie s o f th e 3 4 te s t c h e m ic a ls o n vesicle f o r m a t io n , s p o r u la tio n and m y c e lia l g r o w th o f L a g e n id iu m sp. are p re s e n te d in T a b le 3 . A " n o e f f e c t " do se w as o n e t h a t d id n o t in h ib it d e v e lo p m e n t o f vesicles, z o o sp o re s o r m y c e lia l g r o w th . M y c o s ta tic e ffe c ts o f th e th e ra p e u ta n ts te s te d w e re g ro u p e d in to : A , doses t h a t a llo w e d v e sicle f o r m a t io n b u t n o z o o spores w e re d e te c te d ; B, doses t h a t in h ib ite d b o th ve sicle a n d z o o s p o re d e v e lo p m e n t; a n d C, doses t h a t in h ib ite d th e d e v e lo p m e n t o f th e said se xu a l r e p r o d u c tiv e u n its a n d m y c e lia b u t w h ic h a fte r 4 8 h o u rs a llo w e d m y c e lia to c o n tin u e to g ro w . M y c o c id a l doses are th o s e t h a t k ille d th e fu n g u s as e vid e n c e d b y th e absence o f h y p h a l d e v e lo p m e n t even a fte r seven d a y s in c u b a tio n u n d e r o p tim a l c o n d itio n s .
I t is a p p a re n t f r o m th e d a ta t h a t f u n g it o x ic e ffe c ts o f th e te s te d c h e m ic a ls re s u lte d in in h ib it io n o r r e d u c tio n o f e ith e r vesicles fo r m e d , m o tile z o o s p o re s , o r h y p h a l g e r m in a tio n . A n in te re s tin g o b s e rv a tio n is t h a t, w it h m o s t o f th e c h e m ic a ls te s te d , th e lo w e s t d o se t h a t repressed h y p h a l g r o w th d id n o t n e ce s s a rily in h ib it se xu a l r e p r o d u c tio n .
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T a b le 1 . M y c e lia l g ro w th o f L a g e n id iu m sp in d iffe re n t m edia a fte r 4 8 hours in c u b a tio n .
C u ltu r e M e d iu m
M y c e liu m size ( m m ) a
F 1 6 —3 0 b F 1 1 1 - 2 2 c
N u tr ie n t agar ( N A ) 12 9
B ra in H e a rt I n fu s io n ag ar ( B H IA ) 8 5
P e p to n e -y e a s t e x tra c t-g lu c o s e agar (P Y G A ) 6 6
S a b o u ra u d a 's d e x tro s e agar (S D A ) 0 0
a M ea n o f th re e re p lic a te s
b F v a lu e : 1 0 .4 3 ( s ig n ific a n t a t 1 % le ve l)
c F v a lu e : 9 . 9 6 ( s ig n ific a n t a t 1% le vel)
P o ta s s iu m p e rm a n g a n a te an d m e th y le n e b lu e a ffe c te d b o th z o o s p o re release and h y p h a l g r o w th o f is o la te F 1 6 - 3 0 a t th e least in h ib it o r y doses o f 10 m g /l and 0 .5 m g /l, re s p e c tiv e ly . E c o n a z o le n itr a te y ie ld e d s im ila r e ffe c ts w i t h is o la te F 1 1 1 -2 2 a t 0 .5 m g /l. A p a tte r n o f p ro g ressive r e d u c tio n in th e re la tiv e n u m b e r o f s w im m in g zo o s p o re s a n d in th e size o f h y p h a l d e v e lo p m e n t w a s o b s e rv e d as th e d o se in c re a s e d . T h is p a tte r n w as c o n s is te n tly seen in te sts o n t r e fla n , m e th y le n e b lu e , h y d ro g e n p e r o x id e , c lo tr im a z o le , fu ra n a c e , m a la c h ite g re e n , ba sic fu c h s in , b e n - la te , c a lc iu m h y p o c h lo r ite , p o ta s s iu m p e rm a n g a n a te and fo r m a lin (T a b le 3 ). R e la tio n s h ip s b e t w e e n d o se le ve l an d th e in h ib it io n ra te (%) e s tim a te d b y th e least squares reg ressio n a n a lysis re ve a le d lin e a r r e la tio n s h ip s in b e n la te , c a lc iu m h y p o c h lo r ite , p o ta s s iu m p e rm a n g a n a te , f o r m a lin , b e n z a lk o n iu m c h lo r id e , 2 ,4 -D , e c o n a z o le n itr a te , e th a n o l, d im e th y lfo r m a m id e , p h e n o l, PVP- io d in e a n d re s ig u a rd . O n th e o th e r h a n d , v e ry p o o r a n t im y c o t ic e ffe c ts w e re o b se rve d w it h io d in e , t r ia c e tin , a m p h o te r ic in a n d n y s ta tin . In h ib itio n o f z o o s p o re a n d ve sicle fo r m a t io n b y b o r ic a cid a n d f u n g it o x w e re i n it ia l ly n o te d a t 5 0 0 m g /l a n d a t 10 m g /l b y g r is e o fu lv in . S u p p re s s io n o f m y c e lia l g r o w th b y th e la tt e r c h e m ic a ls was p r a c tic a lly n il. L a s tly , a f lu c t u a t in g e ffe c t o n th e size o f m y c e lia l g r o w th w a s d e m o n s tra te d b y p im a r ic in a lth o u g h zo o s p o re s w e re in h ib ite d at doses o f 5 0 m g /l a n d 1 0 0 m g /l.
R e s p o r u la tio n o f w a sh e d a n d s u b c u ltu re d te s t fu n g i t h a t d id n o t s p o ru la te d u r in g e x p o s u re t o th e c h e m ic a ls s h o w e d t h a t m a jo r it y o f th e se re co ve re d fr o m repressive a c tio n o f th e d ru g . F 1 6 - 3 0 is o la te s e xp o s e d t o e th a n o l, b a sic fu c h s in , a c e tic a cid a n d m a la c h ite gre e n , h o w e v e r, s h o w e d n o r e p r o d u c tiv e responses p o s s ib ly d u e t o p e rm a n e n t da m a g e b y th e se c h e m ic a ls . D a c o n il and P V P -io d in e seem ed t o have e q u a l e ffe c ts o n tre a te d c o lo n ie s o f F 1 1 1 -2 2 .
T a b le 2 . F u n g ito x ic a c tiv ity o f d im e th y lfo rm a m id e and e th a n o l on vesicle fo rm a tio n ( V ) , m o tile zoospores (Z ) and m y c e lia l g ro w th in h ib itio n (M G ) d o f L a g e n id iu m sp.
C o n c e n tr a tio n ( m g / l)
S tr u c tu r e a ffe c te d
D im e th y lfo r m a m id e E th a n o l
F 1 6 - 3 0 F 111 —2 2 F 1 6 - 3 0 F 1 1 1 - 2 2
V + + + + + + +
Z + + + + + + + + + +
M G 0 0 0 0
5 0 0
V + + + + + + + + + + +
Z + + + + + + + + + + + +
M G 0 0 0 0
1 0 0 0
V + + +
Z + + + + + —
M G 0 0 0 0
5 0 0 0
V + +
Z + + + + — —
M G 0 0 0 0
1 0 0 0 0
V + +
Z + + — —
M G 0 14 0 7
5 0 0 0 0
V – –
Z - - * *
—
M G 8 6 4 8 1 0 0 4 5
d — m ean o f th re e re p lic a te s
* * — w a sh e d and s u b c u ltu re d m y c e lliu m d id n o t re s p o ru la te .
A m o n g th e s y s te m ic a n tib io tic s te s te d , a m p h o te r ic in and n y s ta tin e x h ib ite d v e ry p o o r m y c o s ta tic e ffe c ts . T h is m a y im p ly t h a t L a g e n id iu m s p . c e lls ha ve n o s te r o l- c o n ta in in g m e m b ra n e in w h ic h the se c o m p o u n d s c o u ld in d u c e adverse cha ng es (G a le , 1 9 7 8 ). G r is e o fu lv in a n d fu ra n a c e e x h ib ite d lo w levels o f m y c o s ta tic doses. T h e la tte r seems p r o m is in g be cau se o f its a c c o m p a n y in g a n tib a c te ria l a c tio n (D e lv e s -B ro u g h to n & P o u p a rd , 1 9 7 6 ; S h im iz u & T a ka se , 1 9 6 7 ). O n P. m o n o d o n la rva e , h o w e v e r, fu ra n a c e has been fo u n d t o a d v e rs e ly a ffe c t m o r p h o lo g ic a l d e v e lo p
m e n t (G a c u ta n & L lo b re ra , 1 9 7 7 ).
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Table 3. Mycostatic and mycocidal levels (mg/l) of 34 antifungal agents on Lagenidium sp after 24 hours exposure
Chemical
F 1 6 -3 0 F 11 1-32
"N o effect"
Dose
Mycostaticdose Mycocidal
dose
"N o effect"
dose
Mycostatic dose Mycocidal
A B C A B C dose
Acetic acid, glacial 100 500 100 500
Amphotericin B >100 >100
Benlate 10 50 100 500 10 50 100 500
Benzalkonium chloride 1 5 10 50 0.5 1 5 10 50
Boric acid 100 500 1000 500 1000
Calcium hypochlorite 100 500 100 500
Clotrimazole 0.1 0 .5 -1 5 0.1 0.5 -1 5 - 1 0 50
Copper sulfate 1 5 -1 0 0 500 0.1 0.5 -1 5 -1 0 0
Crystal violet 0.1 0.5 1 0.1 0 .5 -1 5
2, 4 D 1 5 10 -100 500 0.1 0.5 -1 5 -1 0 0 500 1000
Dacomil 1 5 -1 0 5 0 -5 0 0 1 5 -1 0 50 -5 0 0
Detergent 1 5 -1 0 50 1 5 -5 0 100
Dimethylformamide 50000 5000 10000 50000
Econazole nitrate 0.1 0 .5 -5 0 0 1000 0.1 0 .5 -1 0 0 50 0-10 00
Ethanol 1000 5000-10000 50000 500 1000-50000
Formalin 5 10 50 5 10 50
Fuehsin (basic) 1 5 - 1 0 50 1 5 10 50
Fungitox 100 50 0-10 00 50 100-1000
Furanace 0.5 1 5 10 50 1 5 -1 0
Griseofulvin 5 10 50 -1 0 0 0 5 10-1000
Hydrogen peroxide 100 50 0-10 00 10 50. 100
Iodine > 1 0 > 5
Malachite green 0.05 0.1 0.5 1 0.5 0.1 0.5 1
Methylene blue 0.1 0.5 1 -1 0 50 1 5
Nystatin* 1000 500 1000
Phenol 10 50 -1 0 0 0 10 5 0 -100 0
Pimaricin 10 50—> 00 50 100->100
Potassium permanganate 5 10 50 100 5 10 50 100
PVP-Iodine 100 500 1000 50 100-500 1000
Resiguard 1 5 -1 0 50 50 100
Tolnaftate 5 10 -100 0.5 5 - 5 0 50 -1 0 0
Triacetin >100 > 5 0
Treflan-R 0.5 0 .5 -1 0.05 0 .1 -5
Trifluralin 0.001 0.005-0.1 0 .5 -1 0 0.001 0.005-0.1 0 .5 -5
A — dose at which vesicles developed but no motile zoospores were detected.
B — dose that inhibited vesicle formation
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T h e b ro a d s p e c tru m a c t iv ity o f c lo tr im a z o le has b e e n d e m o n s tra te d w i t h C a n d id a a lb ic a n s ( Iw a ta & Y a m a g u c h i, 1 9 7 5 ). P im a r ic in , h o w e v e r, re q u ire d h ig h doses t o b e m y c o s ta tic in th e p re s e n t s tu d y . T h e p o o r a n t im y c o t ic a c t iv ity o f t r ia c e tin m a y be a t t r ib u t e d t o th e absence o f esterases in se a w a te r w h ic h c a ta ly z e s its c o n v e rs io n t o th e a c tiv e ly a n tifu n g a l d e r iv a tiv e .
D is in fe c ta n ts te s te d , i.e ., b e n z a lk o n iu m c h lo r id e , d e te r g e n t ( T id e ) , f o r m a lin , p h e n o l, p o ta s s iu m p e rm a n g a n a te a n d re sig u a rd w e re m y c o c id a l a t lo w c o n c e n tr a tio n s a n d m a y b e c o n s id e re d f o r d is in fe c tio n o f e q u ip m e n t an d fa c ilitie s used in c ru s ta c e a n c u ltu r e . A c e tic a c id , b o r ic a c id , c a lc iu m h y p o c h lo r ite , h y d ro g e n p e r o x id e , io d in e a n d P V P -io d in e r e q u ire d su ch h ig h le ve ls t h a t th e y are n o t e c o n o m ic a lly s u ita b le f o r d is in fe c tio n .
T h e p o o r s p o r u la tio n resp on se o f som e tre a te d is o la te s m a y in fe r p a r tia l o r p e rm a n e n t d a m a g e o f sp o ro g e n e sis in L a g e n id iu m sp. e xp o s e d t o th e se fu n g ic id e s . T h e absence o f c o r r e la tio n b e tw e e n b o t h te s t fu n g i m u s t be re g a rd e d w i t h s k e p tic is m u n t il m o re e x te n s iv e s tu d ie s a re m a d e . A c o n s is te n t o b s e rv a tio n in th e p re s e n t e x p e r im e n t w as th e a p p a r e n tly r e la tiv e s e n s itiv ity o f L a g e n id iu m sp. h y p h a e t o th e te s t fu n g ic id e s even a t v e ry lo w d o se s as e v id e n c e d b y t h e d e g re e o f in h ib it io n rates. T h a t th e se in h ib it io n ra te s a re n o t c o r r e s p o n d in g ly a c c o m p a n ie d b y e ffe c ts o n sp o ro g e n e sis leads t o th e c o n c lu s io n t h a t fu n g a l m y c e lia o f L a g e n id iu m s p . are p r o b a b ly a f fe c te d b y fu n g ic id a l a c tio n even a t v e ry lo w d o se s. A lth o u g h c o m p a r a tiv e s tu d ie s o n m y c o s ta tic and s p o ro s ta tic a c tiv itie s o f a n tifu n g a ls s h o w e d n o e v id e n c e t h a t t h e la tt e r is s tr o n g e r th a n th e f o r m e r , in g e n e ra l, fu n g is ta tic a c t iv it y o f v a rio u s su b sta n ce s seems t o ha ve m o re e f f e c t o n m y c e lia l g r o w th th a n o n g e rm tu b e e m e rg e n ce ( Y a n a g ita & Y a m a g is h i, 1 9 5 8 ) .
A lth o u g h m y c o c id a l levels o f th e c h e m ic a ls te s te d w i l l b e id e a l f o r le th a l t r e a t m e n t o r c o n tr o l o f L a g e n id iu m sp. th e h ig h d o se re q u ire d m a y b e le th a l t o th e h o s t. T h e use o f th e m y c o s ta tic c o n c e n tr a tio n s th e r e fo r e is a p ra c tic a l a p p ro a c h . Based o n t h is s tu d y , c lo tr im a z o le , c ry s ta l v io le t, e c o n o z o le n itr a te , m a la c h ite g re e n , t r e f la n a n d t r if lu r a lin s h o u ld b e c o n s id e re d f ir s t in p r i o r it y sin ce th e se e x h ib it m y c o s ta tic e ffe c ts a t less th a n 1 m g /l d o s e . In a d d itio n , o th e r c h e m ic a ls such as b e n z a lk o n iu m c h lo r id e , f o r m a lin a n d re s ig u a rd m a y b e in c lu d e d be ca u se o f t h e ir r e la tiv e ly lo w c o s t. T re a tm e n ts o f re a rin g w a te r c o n ta in in g la rv a e , a d u lt s h rim p s o r c ra b s , h o w e v e r, s h o u ld b e d o n e o n ly a f t e r p r e lim in a r y to le ra n c e e x p e r im e n ts u s in g a t le ast th e m y c o s t a t ic dose p ro v e t o b e safe f o r th e h o s ts . M y c o c id a l do ses o n th e o th e r h a n d , ca n b e u s e fu l f o r d e te r m in in g d is in fe c tio n doses o f e q u ip m e n t a n d fa c ilit ie s used in re a rin g p ro c e d u re s as w e ll as f o r d e s tro y in g b a tc h e s o f in fe c te d la rvae .
5
L ite ra tu re cited :
D e lv e s -B ro u g h to n D .J ., a n d P o u p a rd C .W . 1 9 7 6 . Disease p ro b le m s o f p ra w n s in r e c ir c u la tin g s y s te m s in th e U .K . A q u a c u ltu r e 7 , 2 0 1 - 2 1 7 .
G a c u ta n R .Q . a n d L lo b r e r a A . T . 1 9 7 7 . E ffe c ts o f fu ra n a c e o n th e zoeae a n d m y s is o f P enaeus m o n o d o n . K a lik a s a n , P h il. J o u r . o f B io l. 6 , 2 6 3 - 2 6 8 .
G a le E .F . 1 9 7 8 . P o ly e n e a n tib io tic s . F r o m S y m p o s iu m p a p e rs o n a n tifu n g a l a n t ib io tic s a n d o t h e r a n tifu n g a l a g e n ts b y A . T r in c i. B r itis h M y c o lo g ic a l S o c ie ty B u lle tin 1 1 , 1 3 6 -1 4 4 .
Iw a ta K . a n d Y a m a g u c h i H . 1 9 7 5 . C lo tr im a z o le (C a n e ste n ) — m o d e o f a c tio n in b ro a d s p e c tru m a n t im y c o t ic C a n e s te n , B a y e r p p . 6 -1 0 .
S h im iz u M . a n d T a ka se Y . 1 9 6 7 . A p o te n t c h e m o th e r a p e u tic a g e n t a g a in st fis h diseases, 6 - h y d r o x y m e t h y l- 2 ( 2 - ( 5 - n it r o - 2 - fu r y l) v in y l) p y r id in e , (P -7 1 3 8 ). B u lle tin o f th e Japanese S o c ie ty o f S c ie n t if ic F is h e rie s 3 3 , 5 4 4 - 5 5 4 .
Y a n a g ita T . a n d Y a m a g is h i S. 1 9 5 8 . C o m p a ra tiv e an d q u a n tita tiv e s tu d ie s o f fu n g i t o x i c i t y a g a in st sp o re s a n d m y c e lia . 6 , 3 7 5 - 3 8 1 .
