EFFECT OF MEDIA ON SPORULATION OF THIELAVIOPSIS BASICOLA AND ITS VIRULENCE ENHANCEMENT BY AN AGROBACTERIUM ISOLATE
Kailash C. SRIVASTAVA 1 & John R. KELLER Department of Biology, Seton Hall University, South Orange, N.J., USA
Abstract
Materials and methods
The virulence of Thielaviopsis basicola towards Cucurbita maxima var. 'True Hubbard' was enhanced by an Agro-
(I) Organisms
bacterium isolate and more acutely by the culture filtrate of the bacterium as measured by the increase in the number of chlamydospores of the fungus and decrease in the wet weight of infected plants.
T. basicola strain ATCC 9091 and an isolated bacterial strain (Agrobaeterium Sp.) were used.
Introduction
Two seeds of squash were sown into each of the 4 pots filled with sterile soil and oats that were infested with T. basicola for 20 days. Thus 8 plants were grown at 25 + 1 ~ for 2 months, after which period, one plant was sacrifised to enumerate the chlamydospores of the fungus. Thus prepared squash strain of T. basicola was isolated from soil using techniques of Yarwood (17), Stover (14) and Papavizas (11) and maintained as stock cultures on the slopes of PDA or in PDB or in casein (1%), mannitol (1%), yeast extract (0.1 ~) plus basal salts broth (CMY) and casein (1%), dextrose (1 ~o), yeast extract (0.1%) plus basal salts medium (CDY).
McCormick (10) was the pioneer to note the increased production of perithecia of Thielaviopsis basicola (T. basicola) when grown in association with Cladosporium fulvum and other fungi. Later studies have shown the virulence enhancement of T. basieola on many hosts by a variety of microorganisms, among them: Asterocystis radieis (8); Aphanomyces enteichus, Bacterium tumefaeiens (16); Pythium and Fusarium culmorum (2); Verticillum albo-atrum (1, 13); Rhizoetonia sp. (6) and Nectria radieicola (3, 10 and 12). Jackson (5) found that the stimulation of T. basicola occurs by the spent media filtrate of a bacterial isolate, only if the mannitol is initially present in the culture medium. In the present investigation, the relationship of this bacterium to the pathogenecity of T. basicola was studied using Cucurbita maxima variety 'true Hubbard' (squash) as a host.
(II] Preparation and maintenance of squash strain of T. basicola
(III) Preparation of bacterial culture filtrate TSA stock cultured bacterium was grown at 28 ~ in 1% casein, 1% mannitol and 0.01% yeast extract broth (CMYB) for 14 days, centrifuged under refrigeration and supernatant filter sterilized.
(IV) Culture of Plants
i Present Address: Dept. T.A.M., C.C.R.T.; F.U.E.L., Caixa postal 2111, 86100, Londfina, P.R.; Brazil.
Surface sterilized seeds of squash were grown in sterilized petri dishes lined with 2 layers of sterile filter paper soaked in sterile distilled water for 2 weeks. Thus obtained seedlings were transferred into specially designed micro-
Mycopathologia 71, 3-7 (1980). 0301-486x/80/0711-0003 $1.00. I~) Dr. W. Junk B.V. Publishers, The Hague. Printed in The Netherlands.
observations on the mycelial proliferation and color of roots were graded on zero to 5 scale. Results
As a function of time, increase in the number of chlamydospores of 7". basicola (Fig. 2), mycelial proliferation (Fig. 3), and darkening of roots occurred (Fig. 4), till the roots were completely black by day 30. With time, a significant increase in the pH of Hoagland's medium was also observed (Fig. 5). There was a decrease in the wet weight of treated plants as compared to the control (Fig. 6). The increase in the number of chlamydospores was highest when T. basicola was associated with the culture filtrate of the bacterium, followed by T. basicola + bacterial isolate, which gave higher number of chlamydospores
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Fig. 1. Apparatus used for growing the plants in vitro, showing a squash plant in Hoagland's medium (HM). O s fernbach flasks of 250 ml capacity (Fig. 1) filled with 200 ml of Hoagland's medium (4) and allowed to grow for 6 days. Then these were grouped into various treatments (Table 1).
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( V) Assessment of Virulence Starting from day 13 after transferring the seedling into the Hoagland's medium (Taken as day zero of the experiments), every sixth day, observations were taken for 42 days on the number of chlamydospores, mycelial proliferation and color of roots by excising 0.5 cm piece of root. Same amount of root was also excised from controls to neutralize the effect of cutting on the wet weight of plants, taken on the last day of experiment. The pH of Hoagland's medium was also measured on day 6 after transferring the seedlings, on day zero of the experiment, and the last day of the experiment. The
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--0
6
12
18
24
30
36
42
NO. OF DAY l ~ Fig. 2. Increase in the numbers of chlamydospores of Thielaviopsis basicola (Tb} as a function of time, (X). Tb alone, (O) . Tb + CMYB, (A). Tb + bacterial filtrate (BF), ([3). Tb + bacterial isolate (BI).
Table 1. Experimental design for growing the Cucurbita maxima var. 'true hubbard' (squash) in Microfernbach flasks. TREATMENTS
P~EPLICATES
.............................................................. 1. H o a g l a n d ' s
medium
alone
2. H o a g l a n d ' s
mediUm
+ CMYBl+
BF 2
3. H o a g l a n d ' s
medium
+ CMYBI+
BI 3
4. H o a g l a n d ' s
medium
+ Tb 4
used
5. H o a g l a n d ' s
medium
+ Tb 4 + CMYB 1
treatment
6. H o a g l a n d ' s
medium
+ T b 4 + C M Y B 1 + BF 2
5 replicates
wer~
in each
7. H o a g l a n d ' s m e d i u m + Tb 4 + C M Y B 1 + BI 3 ............................................................... Total
seedlings
used
35
......................................... 1 CMYB
= 1% casein, salts
2.BF
yeast
extract
+ basal
broth.
= Culture was
l%mannitol,0.01%
. ....................
filtrate
first
grown
3.BI
= 48 h o u r
4.Tb
= Thielaviopsis
culture
in w h i c h for
the b a c t e r i a l
isolate
14 days.
of the b a c t e r i a l
isolate
added.
basicola.
than those observed with T. basicola alone (Fig. 2). A similar increase in the mycelial proliferation (Fig. 3) and the color of roots (Fig. 4) was also observed. The virulence of T. basicola was retarded when it was in association with uninoculated CMYB, as judged by the number of chlamydospores, mycelial proliferation, color of roots and the wet weight of plants (Figs. 2, 3, 4 and 6). The above mentioned observations were statistically significant on the basis of Tuckeys, student-t and F tests. As measured by the loss of water from the flasks, in the infected plants the water absorption was fairly diminished in the following increasing order: (1) 7". basicola + CMYB, (2) bacterium alone, (3) T. basicola alone, (4) T. basicola+ bacterium, and (5) T. basicola+culture filtrate of bacterium. In the infected plants, the bacteria added, with or without T. basicola were localised in 'black spot structures'. On the root lengths used for enumeration of the number of chlamydospores, only 1 to 3 such structures
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o
.<
oo 0
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el O
m0
6
12
18
24
30
36
42
NO. OF DAY Fig. 3. Mycelial activity of Thielaviopsis basieola on 0-5 scale. (X). Tb alone, (O). Tb + CMYB,(A) Tb + BF, (D). Tb + BI.
~
"6
12
18 24 30 36 42 NO. OF DAY Fig. 4. Blackening of roots as a function of stimulation of pathogeneeity of Thielaviopsis basicola. (X). Tb alone, (O). Tb + CMYB, (A). Tb + BF, (I-'l). Tb + BI.
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i.
v
~
6*
v
0
NO.
v
OF
DAY ~
v
42
Fig. 5. Changes in the pH of Hoagland's medium on the day 6 after transferring the seedlings, on days zero and at the termination of experiment (day 42). (-X--X). Control, (-A-A-) HM + B F , ( - D - D - ) H M + B I , ( X X ). H M + T b , ( O O ) HM+,iTb+iCMYB, ( A A). HiM + Tb+ CMYB + BF, ( [] [] ). HM +Tb +CMYB + BI, ( ~' '~' )Original ptt of riM. ......___q were observed in the treatments, where the bacterium was associated with T. basicola, but 3-6 structures were seen when the piant was infected with the bacterium alone. Each of these structures contained 50-200 bacteria. While maintaining the stock cultures of T. basicola in the CMY medium, it was observed that after 3 4 passages the growth and sporulation of the fungus was very slow. When, however, the fungus was transferred to potato dextrose broth (PDB) or casein glucose yeast broth (CGYB) for one passage and reinoculated into the CMY, the growth and sporulation was normal. The CGYB turned out to be the best medium for both growth and sporulation. Therefore, the following scheme was used to maintain the stock cultures. CMY 3 passages ~
~PDB or CGYB 1 passage
Discussion
When Thielaviopsis basicola, the causative organism of black root rot disease of many plants that are important economically, is grown in association with the agrobac-
1
2
3
4
5
6
7
Fig. 6. Decrease in the wet weight of infected plants. (1). Control, (2). HM + BF, (3). HM+ BI, (4). HM+ Tb+ CMYB, (5). HM + Tb, (6). HM + Tb+ BI,and (7). HM +Tb+ BF. terium isolate a significant stimulation, in its pathogenecity to Cucurbita maxima var. 'true hubbard' occurred. The culture filtrate of this bacterium also stimulated the pathogenecity of T. basicola, even to a greater extent than by the bacterium itself. These results are in complete agreement with the results of a previous report done under 'in-vitro' conditions (5). The lesser stimulation by the bacterium itself may be attributed to the fact that the bacteria seem to infect the plant roots and perhaps interfere with the infection of the fungus thereby developing a bacterial-fungal competition for the nutrients as reported by Strezelezuk (15). A review of the earlier literature also shows that several other microorganisms have synergistic behaviour to T. basicola (1, 2, 3, 6, 7, 8, 9, 10, 12, 13, 15 and 16). The observation that the fungal pathogenecity is retarded by the addition of CMYB can be attributed to the excessive concentrations of the growth factors present in
the casamino acids and yeast extract, which may also have inhibited the root growth. The pH changes in the Hoagland's medium may be recognized as a possible effect of excretion of the metabolites of the host, Thielaviopsis basicola and the bagterium.
Acknowledgements We express thanks to Dr. N.D. DeProspo, former Chairman of the Department, for providing financial assistance to one of us (KCS). Sincere thanks are also due to Drs. D. G. Smith and R. N. Strange of the University College, London, England and Dr. C. S. Rao of this Department for going through the manuscript and making fruitful suggestions.
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14. Stover, R.H. 1950. The black root-rot disease of tobacco. I. Studies on the causal organisms, Thielaviopsis basicola. Canad. J. Res., Sect.C, 28: 445-470. 15. Strezelezuk, E. 1966. Studies on the rhizosphere microflora of plants resistant and suspectible to soil-borne diseases. III. Incidence of antagonists of Thielaviopsis basicola and Fusarium oxyspommqini in rhizosphere and non-rhizosphere soil. Acta Microbiol. Polo., 14: 87-100. 16. Trotter, A. 1946. On the presence of root tumors in tobacco plantations in the open field., Ric. Ossvz. Divilg. Fieldport Companiaed Mezzogiorno, X: 65-80. 17. Yarwood, C.E. 1946. Isolation of Thielaviopsis basicola from soil by means of carrot discs., Mycologia, 38: 3 4 6 348.