Interactions between Bacillus species and sclerotia of Sclerotium cepivorum

Sod Biol. Biockem. Vol. 21. NO. 1. pp. 173-176. 1989 Printed in Great Britain. All rights reserved

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0038-0717,89 $3.00 + 0.00 c 1989 Pergamon Press plc

SHORT COMMUNICATION INTERACTIONS BETWEEN ~~C~~~~~ SPECIES AND SCLEROTIA OF SCL~R~TI~~ C~~I~~RU~ D. BACKHOVSEand A. STEWART Botany Department, University of Auckland, Private Bag, Auckland, New Zealand (Accepted

ZS May

Sclerotia of Sclerorium cepivorum Berk.. the cause of A&m white rot, can survive in soil for several years with little loss of viabilitv (Colev-Smith. 1979). Because economicallvviable fun*gihde treatments have had limited success in controlling the disease and do little to reduces inoculum density in the soil, considerable attention has been focused on the search for biological control measures. BaciNus species are often associated with sclerotia of S. cepirorum. Dickinson and Coley-Smith (1970) and ColeySmith and Dickinson (1971) showed that sclerotia exuded substances, especially carbohydrates, which stimulated growth of Bucihs suhtilis (Ehrenberg) Cohn. Utkhede and Rahe (1980. 1983) isolated B. suhfilis from sclerotia collected from several parts of the world. Most of their isolates gave significant protection against white rot when applied to onion seeds at the time of sowing. In Queensland, Wong and Hughes (1986) found that B. Iicheniformis (Weigmann) Chester was the most common antagonistic bacterium recovered from non-viable sclerotia of S. cepirorum. Rahe (1983) suggested that “there is much more than a casual relationship between 8. subtilis and sclerotia of S. cepi~oruni”. The recovery

of Buciihs species from sclerotia which had been surface-sterilized (Utkhede and Rahe, 1980) and which failed to germinate (Wong and Hughes. 1986) suggests that the bacteria can parasitize sclerotia, leading to loss of viability. As part of a study of biological control of onion white rot we wished to determine whether, and under what conditions, such parasitism occurred. An experiment on colonization of sclerotia by B. subrilis in soil had suggested that bacteria were restricted to the surface of the rind. We therefore tested the ability of Bucifhts species to invade sclerotia in r&o, and examined the effects of bacteria on sclerotia with a view to understanding possible mechanisms of the biological control reported by Utkhede and Rahe (1980). All organisms used originated from onion fields at Pukekohe. South Auckland. New Zealand. Two isolates of B. subrilis. 611 from soil and 814 from surface-sterilized sclerotia. were used. Both isolates were strongly antagonistic to S. cepirorum in rho. A weakly antagonistic strain of E. polymyxa (Prazmowski) Mace from surface-sterilized sclerotia was used in some experiments. All bacteria were identified by the methods of Claus and Berkeley (1986). Sclerotia of S. cepicorum were grown on autoclaved wheat grains and harvested by wet sieving. Spores of B. subtilis Bll and 814 were prepared by growing the bacteria in a liquid medium containing 1% glucose, 1% Difco Bacto-peptone. I mM CaClr. 1 rnM MgSQ,, 1mkt MnSO, and 100 PM FeSO, for 5 days at 23’C. Spores were harvested by centrifugation and rinsed twice with distilled water. Pukekohe field soil (clay-loam, pH 7.5) was autoclaved twice at 12l’C for 30min on successive

1988)

days. Autoclaved and non-sterile soils were seeded with 200 sclerotia g-i and SOg lots placed in IOOml screw-top ~lyethylene containers. Spores were added, at 1O’cfu g-i, and the soil moistened to Ileld capacity. Control pots had distilled water only added. Half of the pots were air-dried for 48 h after addition of spores and sclerotia and then remoistened. Three replicates of each treatment were maintained at 20°C for 3 months. Sclerotia were recovered by wet sieving (without sucrose flotation), surface-sterilized for IOmin with 1% NaQCl and plated onto potato dextrose agar (PDA) and held at room temperature (18-23°C). Percent bacterial colonization was determined for 30 sclerotia from each pot. The complete assay was performed on two separate occasions, and a third assay done using 50 sslerotia from each of two pots of each treatment. Results were analysed by analysis of variance or pairwise r-test as appropriate. Twenty xlerotia from which colonies resembling B. subtilis pw were fixed and embedded in glycol methacrylate (GMA) (Backhouse and Stewart. 1987). and sections-were examined for the dist~bution of bacterialcells. To test for ability to grow on sclerotial exudates, a sclerotium extract agar was prepared. Sclerotia were rinsed six times with sterile distilled water, dried overnight over CaCl, (Coley-Smith et al., 1974). and extracted for 24 h at 23’C with IOml distilled water g-’ of sclerotia. Sclerotia were removed by filtration through Whatman No. I paper, agar was added to the extract, and the medium autoclaved. All bacteria were grown on this medium for three successive transfers at 2S’C. and growth compared with that on distilled water agar. The ability of bacteria to invade sclerotia was tested by exposing sclerotia in bacterial cultures in half-strength Difco nutrient- broth. Sclerotia were surface-sterilized for 10 min with 1% NaQCl, rinsed three times with sterile distilled water, and then added either fresh, after drying overnight over Cat&. or after gently grinding with a mortar and pestle, to 24 h cultures of bacteria. In one experiment B. sub&_r 814 was used, and in a repeat experiment all three bacterial isolates were tested. Control sclerotia were incubated in broth containing 50 mg chloramphenicot 1-i to suppress contamination. After 6 days incubation on a rotary shaker at 23’C. sclerotia were fixed. embedded in GMA and sections stained with Coomassie Blue. Toluidine Blue or the periodic acid-SchiIT (PAS) procedure (Backhouse and Stewart. 1987). At least 8 sclerotia from each treatment were examined. Bacteria grew from many surface-sterilized sclerotia from soil pots (Tables I and 2). Recovery rates were very variable between replicate pots, with high standard deviations (Table I). There was no significant (P Q 0.05) effect of air-drying or use of autoclaved soil (Table I). Bacterial recovery rates were significantly (P b 0.05) higher for 814 than for Bl I (Table 2). No bacteria grew from sclerotia exposed to soil

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174 Table I. Eikt

Treatment Auto&wed Non-sterile Air-dried NOtl-dCicd

Short communications of soil treatments on recoveryof B. mbrilis Bl4 from sclcrotia Percent sclerotia from which B. subalis greW” Assay I Assay 2 27.2 (19.8) 20.0 (7.0) 25.0(16.3) 22.2it4.3j

13.9 (9.3) 18.3 (14.0) 16.7114.0) 15.6 ig.6)

‘Mean (and SD). n = 6. bEtTects of treatments and interactions between treatments not significant at P = 0.05 (analysis of variance).

without added spores. The third assay gave significantly (P c 0.05) higher recovery rates than the first two assays for both bacteria (Table 2). When sclerotia from which bacteria grew were sectioned, bacteria were found to be restricted to the outside surface (Fig. I). No bacteria were found inside any of the 20 sclerotia examined. All bacterial isolates grew on sclerotium extract agar through three successive transfers but showed negligible growth on water agar. When sclerotia were added to liquid cultures of bacteria, bacteria were found to enter sclerotia only when the rind was damaged. No differences were seen between air-dried and undried scierotia in their response to any bacterium. In the absence of bacterium, between I and 5% of intact sclerotia in each flask were seen to have germinated by production of mycelium. Most remained ungerminated, and in section non-germinated sclerotia resembled the resting sclerotia described by Backhouse and Stewart (1987). All damaged sclerotia held in nutrient broth alone germinated to produce spherical, hyaline colonies up to 2 mm dia. Cultures of B. poltm.rxo frequently became contaminated with other bacteria, presumably introduced onto sclerotia during handling, but this did not affect their usefulness for comparison with B. subrilis. Intact sclerotia exposed to cultures of f?. pol.rm_r.ru behaved similarly to those incubated in nutrient broth alone. A small proportion of sclerotia had germinated, but most showed no sign of morphological change. In section nongerminated sclerotia resembled control sclerotia. Hyphal contents stained strongly, and numerous protein bodies were seen (Fig. 2). All damaged sclerotia germinated (Fig. 3). Germinative hyphae in the presence of B. polymys~~were pigmented and limited in growth, so that colonies did not form. In section, hyphal reserves were seen to be depleted by germination, but distinct protein bodies were still present in some cells (Fig. 3). Bacterial cells were often seen inside sclerotia in areas where the rind had been ruptured. The presence of bacteria did not appear to affect the germination process. There was no obvious difference in the effects of BI I (isolated from soil) and 814 (from sclerotia) on sclerotia. No germination was seen in any sclerotium incubated in cultures of B. subfilis. No bacteria were seen inside intact sclerotia. Medullary hyphae of undamaged sclerotia stained strongly with coomassie blue, and protein bodies were present. However, protein bodies were indistinct in sections stained with Toluidine Blue (Fig. 4). indicating that they had undergone some change. Cells of B. subfilis were common inside damaged sclerotia (Figs 5 and 6). The bacteria appeared to penetrate deeply only where the sclerotium was cracked. Where the rind had been abraded away but the medulla was otherwise undamaged invasion was limited to surface cells. No erosion of cell walls or extracellular polysaccharide was detectable in sections stained with PAS. Most hyphae contained material that stained with Coomassie Blue (Fig. 5) but cell contents were irregular in outline and often indistinct. Almost all material stainable with Toluidine Blue had been

extracted from sclerotia when B. subdis was present within them (Fig. 6). The experiment in which sclerotia were incubated in soil containing bacterial spores suggested that bacteria were limited to the external surface of sclerotia. Colonies of B. subrilis that developed when sclerotia were plated onto PDA presumably originated from spores adhering to the rind that survived hypochlorite disinfection. Whether the higher yields of B14, originally isolated from sclerotia. were due to a greater ability to colonize sclerotia or higher tolerance to hpochlorite could not be determined. The only significant effect on bacterial recovery besides the isolate of B. sub&s used was variation between assays. Vimard er (11.(1986) found that temperature affected rates of bacterial contamination of sclerotia of S. cepiuorum. Many other factors influencing dislodgement of spores, efficiency of surface-sterilization, and spore germination could also account for this variation. If the bacteria had been present within the sclerotial tissue, less variability in recovery rates between assays might have been expected. All bacteria tested could grow on sclerotial exudates. The surface of sclerotia from soil is rough. with many collapsed cells (New er al., 1984: Backhouse and Stewart, 1987) which would provide niches in which bacteria could become lodged. Sclerotia are therefore well suited to surface colonization by microorganisms. As well as Gram-positive bacteria, Leggett and Rahe (1985) found Gram-negative bacteria and a variety of fungi on non-sterilized sclerotia. The preponderance of Bacillus species, at least among antagonistic bacteria, isolated from sclerotia in the studies of Utkhede and Rahe (1983) and Wong and Hughes (1986) probably reflects the the resistance of their spores to surfacesterilization and other treatments used. On the other hand, bacteria do occur inside some sclerotia in soil (Backhouse and Stewart, 1987). Since Bacillus species could not invade intact sclerotia, these bacteria must enter through openings in the rind caused by other agents. There have been several reports of enhanced decay of sclerotia of S. crpivorum caused by drying and subsequent rewetting (Smith, 1972; Leggett rr (II., 1983; Leggett and Rahe. 1985). However, air-drying of sclerotia did not cause sufficient damage to the rind to allow even actively growing cultures of bacteria to penetrate. The effect of drying must therefore be enhancement of microbial growth due to nutrient leakage as Smith (1972) suggested, rather than weakening of the sclerotium. Raised nutrient concentrations due to drying would be only transient (Coley-Smith er al., 1974). Decay would thus depend on the presence of suitable hyperparasitic fungi (not bacteria) at the time of drying. Differences in soil microflora could account for the apparent lack of effect of drying on survival reported by Coley-Smith Ed al. (1974) and Papavizas (1977). Bacillus subrilis was able to inhibit germination of sclerotia in cifro and to kill sclerotial hyphae. These effects may have been due to toxin production. rather than competition for nutrients or O?. since sclerotia were able to germinate in the presence of active cultures of B. polymJ.ro. Changes were also seen in the staining reactions of hyphae of intact sclerotia incubated in B. subriliscultures, implying that some B. subrilis toxins can diffuse through sclerotial rind. The significance of these effects on germinability in the field

Table 2. Recovery of bacteria from sclerotia incubated 3 months in soil containing spores of 8. rubrilis Bl I and BIJ Assay I 2 3

Percent sclerotia from which E. suhrilis grew’ Control B14 BII 0 ab Oa Oa

Oa 0.6 a 5.5 b

23.6~ 16.1 c 45.0 d

‘All treatments and replicates for each bacterium combined. %Jalueswith same letter do not differ at P = 0.05 (r-test).

Short communications

Fig. 1. Section of sctrrotium recovered from soil, from which B. u&f& grew, stained with lofuidine Blue. No bacteria are present inside the sclrrotium. Figs 3 and 3. Sections of sclerotia incubated in cultures of B. ~~~~~~.~u. Fig. 2. Intact sclerotium. Toluidine Blue stain. Numerous well-defined protein bodies occur in medullary celb. Fig. 3. Damaged sclrrotium, Coomassie Blue stain. Germination occurs despite the presence of bacteria in the medium. Figs 4-6. Sections of sclerotia incubated in cultures of It. subtilis. Fig. 4. intact sclerotium. Totuidine Biue stain. Hyphal contents are well organised but staining of protein bodies is reduced (cf. Fig. 2). Fig. 5. Damaged sclerotium. coomassie btur stain. Bacteria (arrows) have entered through crack in rind. Fig. 6. Damaged sclerotium containing bacteria, Toluidine Blue stain. Most stainable material has been lost.

Short communications

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could not be tested since we cannot achieve suBlciently reproducible host-induced germination. However, under conditions particularly favourable for bacteria1 growth some inhibition of germination might occur and would account at least in part for the reports of successful. biological control using B. subtilis (Utkhede and Rahe. 1980, 1983). It seems unlikely that the frequent recovery of Bacillus species from sclerotia of S. cepicorum is due to a special relationship. Rather, their association depends more on the remarkable biology of Bacillus as an opportunistic colonist, antibiotic producer and sporeformer. REFERENCES

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