Development of a Methodology for the Production of Aphanomyces cochlioides
Test 1 Test 2 4.20
5.00 5.50 5.70 6.0 6.20 6.30 6.40 6.50 6.60 6.75
- 0 0.5 d
- 11.6 b
- _ - 18.3a
- -
0.4 e -
1.3 e 8.2 d 15.5 c 22.4 b 28.9 a
- - 29.2 a
-
46.0 a 41.7a 47.0 a 50.7 a
1Small letters indicate Duncan's multiple range groupings of treatments that do not differ significantly at the 5% level.
2Results expressed as means of 2 experiments, with 4 replications of each treatment.
3Results based on counts of spore suspensions from each of 3 cultures/treatment.
The effect of light
In separate experiments, oatmeal homogenate agar plate cultures
of isolate Ac-9 were exposed to three different light intensities and
exposure periods during a 15-day period beginning immediately after
inoculation of the plates. In two experiments growth chambers were
maintained at 23°C and provided with fluorescent illumination. In a
third experiment, cultures in a windowless room were exposed to
intermittent light from a ceiling fluorescent lamp. Light intensities
ranged from 60 to 1 500 foot candles and daily exposure periods from
3.6 hours (average) to 24 hours. Spore production in the cultures
exposed to the three light treatments was compared with that of cul-
tures incubated within adjacent metal canisters that excluded light. In
each e x p e r i m e n t light adversely affected o o s p o r e p r o d u c t i o n
236 JOURNAL OF THE A. S. S. B. T.
(Table 4). C u l t u r e s exposed to a relatively low light intensity (60 foot candles) a n d short e x p o s u r e p e r i o d (3.6 hr) p r o d u c e d a p p r o x i m a t e l y one-half the n u m b e r of oospores of cultures in d a r k n e s s . C u l t u r e s exposed to h i g h e r light intensities a n d l o n g e r e x p o s u r e s p r o d u c e d negligible n u m b e r s of spores. T h e s e results confirm those of Fowles (4) c o n c e r n i n g the inhibition of sexual r e p r o d u c t i o n in A. cochlioides a n d A. stellatus by light.
Table 4.—Effect of light on production of oospores by Aphanomyces cochlioides in plates of oatmeal homogenate agar.
Light intensity and daily exposure period 1) 400 foot-candles; 24 hr
Unilluminated 2) 1500 foot-candles; 14 hr
Unilluminated
3) 60 foot-candles; 3.6 hr (average)2
Unilluminated
No. oospores/field1
0 95.3
4.3 a 109.0 b
57.0 a 104.0 b
1Results based on counts in each of three plates/treatment. Small letters indicate Duncan's multiple range groupings of treatments that do not differ significantly at the 5% level.
2Illumination period ranged from 0 to 8 hr/day, and totaled 50 hr for the 14-day period.
Viability of oospores produced in vitro
Because o u r ultimate objective in o b t a i n i n g o o s p o r e s in vitro was to use t h e m as inoculum, we accordingly tested their ability to ger- m i n a t e a n d to initiate infection. G e r m i n a t i o n tests were c o n d u c t e d with oospores of isolate Ac-7 that h a d b e e n p r o d u c e d in c u l t u r e a b o u t 5 m o n t h s previously a n d h a d b e e n a p p l i e d s u b s e q u e n t l y i n liquid suspension to microscope coverslips to dry. T h e coverslips were placed in Van T i e g h e m cells w h e r e t h e a d h e r e n t oospores were exposed to h a n g i n g d r o p s of various solutions c o n s i d e r e d as possible g e r m i n a t i o n substrates. T h e spores were observed intermittently d u r i n g a 60 day p e r i o d . After the 14th day a n d 36 days thereafter, 12 oospores a m o n g t h e estimated 1 . 9 x l 04 on t h e 21 coverslips a p p e a r e d to g e r m i n a t e . Each p r o d u c e d a g e r m h y p h a similar in a p p e a r a n c e to those of Aphano- myces euteiches as d e p i c t e d by various a u t h o r s (8, 13) but n o n e a p p e a r e d
to function as zoosporangia. T h e m a x i m u m observed l e n g t h of any of t h e g e r m h y p h a e m e a s u r e d a b o u t 200 μ, b u t t h e length of most was considerably s h o r t e r . T h e n u m b e r o f spores g e r m i n a t i n g p e r esti- m a t e d n u m b e r of spores e x p o s e d to each solution was: casein hydroly- sate (30-600 mg/1), 6/5,400; c o c o n u t milk (1-15%), 0/7,200; indole acetic acid ( 1 0- 4m g / l ) , 0/1,000; n a p h t h a l e n e acetic acid ( 3 x 10- 2mg/l), 0/1,800; soil leachate, 4/1,800; sugarbeet seedling decoction (20 g/1) 3/1,800.
VOL. 17, No. 3, APRIL 1973 237
Viability of in vitro -produced oospores was demonstrated also in infection tests. Sugarbeet seedlings were grown in pots of soil infested with oospores of isolate Ac-7 when the seeds were planted. In one experiment, parts of dried mycelial mats from liquid cultures with oospores at various density levels were buried just below the soil surface in the center of each pot. T h e estimated number of spores/15 mm square portion of mat ranged from 0 to 4x 10
4. Around each buried part of mat, 20 seed balls were planted at a distance of about 20 mm. Within 15 days after planting, some seedlings in each pot infested with 10
4or more oospores developed typical symptoms of Aphanomyces seedling blight. Final disease evaluations made 30 days after planting showed a definite association between oospore density and disease incidence (Table 5). No plants appeared to be infected in pots that received .5x 10
4or less oospores. In a similar experiment, three replicate pots were infested with oospores in dried suspensions on microscope coverslips at densities of approximately . 1 , .2, .3, .7, 1.3, and 2.6 X 10
4/4-in. pot. Disease incidence increased with an increase in oospore density from .7 to 2.6x 10
4/pot; no infection was noted in pots with fewer than .7x 10
4spores.
Table 5.—Incidence of sugarbeet s e e d l i n g blight in pots of soil implanted with dried mycelial mats of Aphanomyces cochlioides containing different numbers of oospores.
Est. N o . oospores/pot (x 104)
3.3-4.5 1.0
.2- .5 0
0 (no mycelial mat)
N o . pots with s e e d l i n g s Blighted/Infested
4/4 1/1 0/4 0/4 0/4
Avg. N o . seedlings pot Blighted/Emerged
32.8/33.8 27.0/27.0 0/40.3 0/37.0 0/38.0
T h e r e is evidence that oospores may remain viable for relatively long periods. Inoculum was prepared by grinding oatmeal broth cultures rich in oospores in a blender, mixing the resultant suspension with vermiculite (1:2.5 parts, V:V) and drying it thoroughly. This inoculum was stored for 18 months and then applied at the rate of approximately 9 x l 0
4spores/4-in. pot. A high incidence of seedling blight subsequently developed.
Discussion
From the results of this study, a methodology form vitro produc-
tion of A. cochlioides oospores has been established. Oatmeal homog-
enate broth has been the medium of our choice because it can be
238 JOURNAL OF THE A. S. S. B. T.
p r e p a r e d m o r e easily a n d m o r e c o n v e n i e n t l y t h a n most o f t h e o t h e r m e d i a yielding large n u m b e r s o f o o s p o r e s . O o s p o r e yields e x c e e d i n g 2 . 0 x l 04/ m l have b e e n consistently o b t a i n e d i n . 5 % o a t m e a l h o m o g - e n a t e b r o t h adjusted t o p H 6.5 a n d with c u l t u r e s i n c u b a t e d i n d a r k n e s s . With o a t m e a l m e d i u m , t h e r e is a p p a r e n t l y no a d v a n t a g e in u s i n g a r e p l a c e m e n t m e d i u m unless acceleration of s p o r u l a t i o n is d e s i r e d . H o w e v e r , with a substrate such as YS chemically defined m e d i u m , t h e use of a r e p l a c e m e n t m e d i u m such as well w a t e r is r e c o m m e n d e d as a m e a n s of significantly increasing t h e yield of o o s p o r e s .
T h e s p o r e g e r m i n a t i o n tests a n d t h e infection tests indicate t h a t t h e o o s p o r e s p r o d u c e d in vitro a r e viable. T h e relatively low g e r m i n a - tion r a t e (ca 1:1,600) o b s e r v e d in o u r artificial substrates may n o t necessarily r e p r e s e n t t h e g e r m i n a t i o n r a t e of s p o r e s in a m o r e n a t u r a l e n v i r o n m e n t of field soil with host p l a n t s . H o w e v e r , t h e n u m b e r of s p o r e s r e q u i r e d to initiate e x p e r i m e n t a l infection of relatively large n u m b e r s of seedlings can be p r o d u c e d readily in vitro. With a typical yield of 2.5 X 104 o o s p o r e s / m l , o n e liter of o a t m e a l h o m o g e n a t e b r o t h yields e n o u g h spores to inoculate 1,250 four-inch pots of soil at a r a t e of 2 . 5 x 104 spores/pot.
In c o n s i d e r i n g the use of o o s p o r e s p r o d u c e d in vitro as i n o c u l u m t h e i r r e t e n t i o n of viability in s t o r a g e for p e r i o d s e x c e e d i n g 1 y e a r is p e r t i n e n t . F u r t h e r m o r e , they can be a p p l i e d readily to t h e soil in c o n t r o l l e d quantities w h e n i n c o r p o r a t e d with a c a r r i e r such as ver- miculite. F u r t h e r studies to d e t e r m i n e t h e feasibility of using o o s p o r e s for e x p e r i m e n t a l inoculation i n t h e g r e e n h o u s e a n d f i e l d a p p e a r t o b e w a r r a n t e d .
S u m m a r y
T h e most favorable m e d i a for p r o d u c t i o n of Aphanomyces cochli- oides o o s p o r e s in vitro ( 1 0 0 + spores/low p o w e r field) w e r e o a t m e a l , b u c k w h e a t groats, p e a r l e d barley, a n d s u g a r b e e t seedlings. T h e use o f a r e p l a c e m e n t m e d i u m accelerated o o s p o r e p r o d u c t i o n a n d , with s o m e m e d i a , also increased t h e n u m b e r o f o o s p o r e s . O o s p o r e p r o d u c - tion at initial pH values of 6.30-6.75 was significantly g r e a t e r t h a n at l o w e r a n d a t h i g h e r p H . L i g h t i n h i b i t e d o o s p o r e p r o d u c t i o n . O o s p o r e s p r o d u c e d in vitro w e r e able to g e r m i n a t e a n d initiate infec- tion of s u g a r b e e t seedlings in t h e g r e e n h o u s e .
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