INTRODUCTION

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Possession of chitinase genes is widespread among many taxa of nonfilamentous soil bacteria (11). Yet several of these bacteria have been shown to be poor degraders of chitin in soil or soil-like model systems, due to their inability to penetrate chitin particles (7, 12). Filamentous microorganisms, such as fungi and actinomycetes, and gliding bacteria are much more efficient in degradation of chitin particles (7, 10, 12). Indeed, the decomposition of chitin in terrestrial soils appears to be attributable mainly to fungi and actinomycetes (7, 10). The continued maintenance of chitinase genes by many nonfilamentous bacteria, however, strongly suggests that they confer some selective advantage on the cells harboring them.

Chitin is an important constituent of the cell walls of most fungi (3), and chitinolytic bacteria have received considerable attention as potential biocontrol agents due to their ability to lyse hyphae of fungal crop pathogens (4, 14, 23). Chitinases of some mycolytic bacterial strains have been shown to destabilize cell walls of fungal pathogens (4, 15). Hyphal tips appear to be especially susceptible to the lytic activities of chitinolytic bacteria.

The ability to lyse the tips of fungal hyphae may allow nonfilamentous chitinolytic soil bacteria to utilize living fungal hyphae as an additional growth substrate (5). This could be an important selection pressure for the maintenance of chitinase genes, given the limited availability of growth substrates in soils (25). There is, however, only limited information on the population dynamics of chitinolytic bacteria during mycolytic activities. This is because most studies have been done using either high densities of chitinolytic bacteria or media that, in addition to hyphae, contain other nutrients supporting bacterial growth (2, 15, 19). In the few cases in which minimal liquid media were used, pretreatment (homogenization or partial fragmentation during washing steps) and age (autolysis) of precultured hyphae may have resulted in leakage of hyphal contents and, consequently, stimulation of bacterial growth and mycolytic activity (13, 16, 23). Hence, it is not clear whether mycolytic growth can be initiated by chitinolytic soil bacteria under natural conditions, i.e., when they are exposed to intact growing fungal hyphae in soil in the absence of other nutrients.

The first aim of this study was to monitor the dynamics of nonfilamentous chitinolytic soil bacteria during exposure to growing fungal hyphae under nutrient-limited conditions in a soil-like system. The second aim was to address the role of chitinase in the response of bacterial populations to the presence of fungal hyphae.

	MATERIALS AND METHODS

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Bacterial strains. The strains used in this study were selected from a collection of chitinolytic bacteria isolated from sandy coastal dune soils in the Netherlands (5). Analysis of whole-cell fatty acid methyl ester profiles (MIDI-FAME) and general cell and colony morphology had revealed that Pseudomonas spp., Stenotrophomonas spp., and Cytophaga spp. were the most commonly isolated nonfilamentous, chitinolytic bacteria in these soils (4), and representatives of each of these bacterial groups were chosen for experimental use. Amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), as well as denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments (17), revealed a high level of relatedness between the chitinolytic pseudomonads chosen for this study (W. De Boer, P. J. A. Klein Gunnewiek, and G. A. Kowalchuk, unpublished data). Nearly full-length 16S rDNA sequences (±1,450 bp) were determined for two of these strains (EMBL accession numbers AJ310394 and AJ310395). Sequence comparison against known 16S rDNA sequences (1) revealed that these bacteria were not true pseudomonads but rather comprised a group within the -subclass of the Proteobacteria, with 94 to 95% sequence identity to isolates within the genera Herbaspillum, Matsuebacter, Janthinobacter, and Ultramicrobacterium. The sequences of the two strains examined showed 98% sequence identity with each other. Therefore, in this article we will refer to this group as unidentified chitinolytic -subclass Proteobacteria, or CPs. The number of chitinolytic strains selected were 9, 8, and 3 for the CPs, stenotrophomonads, and cytophageles, respectively. Preliminary experiments had shown that the CPs and stenotrophomonads were slow and inefficient degraders of chitin particles in soil microcosms, whereas the Cytophaga-like strains were fast and efficient chitin degraders (7; W. De Boer and P. J. A. Klein Gunnewiek, unpublished data). In addition, eight common strains of nonchitinolytic dune soil bacteria, belonging to the genera Pseudomonas, Burkholderia, and Comamonas, were selected for control experiments.

Fungal strains. The study was conducted using three filamentous fungal species that are abundant in coastal dune soils, namely, the ascomycete Chaetomium globosum, the hyphomycete Fusarium culmorum, and the zygomycete Mucor hiemalis (8).

Bacterial dynamics during mycelial development in sand. Chitinolytic bacterial isolates were grown on chitin-yeast agar (CYA) at 20°C for 14 days. Medium composition was as described by De Boer et al. (7) but with the addition of 0.01 g of yeast extract (Difco, Detroit, Mich.) liter¹. Nonchitinolytic bacteria were grown on 10-fold-diluted tryptic soy broth agar (TSBA) at 20°C for 7 days (7). Fungi were grown on potato-dextrose agar (PDA) at 20°C for 2 (Mucor and Fusarium) or 7 (Chaetomium) days (6).

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Effect of allosamidin on bacterial dynamics. Allosamidin is a powerful inhibitor of endochitinases, previously detected in the mycelial extract of a Streptomyces sp. (21). A stock solution of allosamidin (Eli Lilly and Company, Indianapolis, Ind.) was made in 5 mM acetic acid (22). Preliminary tests showed that allosamidin (10 µM) was an effective inhibitor of chitinase activity for the bacteria used in this study, both in sand and on agar media. Mycelial extension of the three fungi used was not affected (De Boer and Klein Gunnewiek, unpublished results).

Microscopic observations. Hyphae were picked aseptically from sand containing a mixture of the CPs and were fixed by heat on glass slides. A drop of sterile, demineralized water containing 2 µg of 4',6-diamidino-2-phenylindole (DAPI) (Sigma Chemical Co., St. Louis, Mo) liter¹ was put on top of the hyphae. After 5 min of incubation in the dark, the slides were rinsed with sterile demineralized water. Excess water was removed with filter paper. A cover glass was mounted with a drop of antifade solution (24) and sealed with clear nail polish. Microscopic examination of the hyphae for the presence of bacteria was done under UV excitation using a Leitz epifluorescence microscope.

Data analysis. Data were analyzed by means of analysis of variance (ANOVA). Where necessary, log transformations were applied to data sets in order to establish homogeneity of variances. Differences between means were inspected using Tukey's honestly significant difference at the 5% level.

Nucleotide sequence accession numbers. The 16S rDNA sequences determined in this study have been deposited in EMBL under accession numbers AJ310394 and AJ310395.

	RESULTS

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Response of bacterial groups to mycelial development in sand. During the 1-week preincubation in sand, bacterial numbers had increased from 10⁴ to about 10⁵ CFU g of sand¹ for all bacterial groups tested. The introduction of fungi resulted in a strong increase in the number of CPs in the mycelial zone, whereas the numbers of nonchitinolytic bacteria were not significantly affected (Fig. 1C and D). Two weeks after introduction of the fungi, the growth stimulation of the CPs was already detectable, especially in the Mucor zone. FAME-gas chromatography (GC) analysis and morphological inspection of colonies revealed that this growth stimulation was not restricted to a single strain. The other two chitinolytic groups, namely, cytophageles and stenotrophomonads, were only slightly, albeit significantly, stimulated by the presence of fungi (Fig. 1A and B).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Response of chitinolytic and nonchitinolytic dune soil bacteria to invading hyphae of the fungi C. globosum and M. hiemalis in purified sand. CFU of the different bacterial groups in the mycelial zone and in a comparable zone without fungal inoculum were determined 2 and 6 weeks after introduction of the fungal inoculum (PDA disks on glass slide). Data represent the means and standard deviations for six replicates that were harvested at the indicated times.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Temporal dynamics of chitinolytic -subclass Proteobacteria in purified sand in the mycelial zones of C. globosum and M. hiemalis. Arrows indicate the times when the mycelial extension stopped. Data represent means and standard deviations for four replicates that were harvested at the indicated times.

Effect of allosamidin on dynamics of CPs in mycelial zones. In the sand containing allosamidin, the CPs still increased in number in response to the presence of each of the three fungi tested (Fig. 3). However, without addition of allosamidin, this bacterial increase was significantly greater in the mycelial zones of Chaetomium and Fusarium. In contrast, allosamidin had no significant effect on the CPs in the mycelial zone of Mucor.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Effect of the chitinase inhibitor allosamidin (10 µM in soil solution) on proliferation of chitinolytic -subclass Proteobacteria in the mycelial zones of three fungi in purified sand. Data represent means for four replicates that were harvested 4 weeks after introduction of the fungal inoculum (PDA disks on stainless steel disks). Striped bars, allosamidin treatments; solid bars, controls. Statistical significant differences (P < 0.05) between allosamidin and control treatments are indicated by asterisks.

Microscopic observations. Bacterial aggregates were attached to many hyphae picked from sand containing the CPs, as observed by fluorescent microscopy (Fig. 4), and this was true for all three fungi tested. In several cases, hyphal tips were completely covered by bacteria (Fig. 4A). Occasionally, crimping of such tips was apparent. Bacteria were, however, also found to be attached to other parts of hyphae (Fig. 4B). Addition of allosamidin reduced the numbers of attached CPs, but this was not quantified.

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Microscopic observations of DAPI-stained fungal hyphae that were picked from sand containing chitinolytic -subclass Proteobacteria. (A) A hyphal tip of F. culmorum covered by bacteria. (B) Separate clusters of bacteria along a hypha of F. culmorum. Scale bar, 10 µm.

	DISCUSSION

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Several studies have dealt with the effects of chitinolytic soil bacteria on plant-pathogenic fungi, in the context of demonstrating their potential as biocontrol agents. These studies have involved applications of high densities of bacteria, use of nutrient-rich liquid or agar media, or addition of hyphal fragments (15, 16, 19). Under such conditions, many chitinolytic soil bacteria were observed to lyse fungal hyhae. In addition, several studies have demonstrated that purified chitinase of potential biocontrol strains can cause deformation of living hyphae (18, 26). However, from these studies it is not clear whether chitinolytic bacteria are also able to attack living fungi under natural conditions, i.e., at low cell densities in the soil.

This study showed that chitinolytic -subclass Proteobacteria (CPs) isolated from coastal dune soils were able to grow at the expense of living fungal hyphae in sand without the presence of other carbon sources. They could do so at cell densities that are common for dune soils (5, 7). Both pathogenic and saprophytic dune soil fungi stimulated the growth of the CPs, suggesting that their growth on living fungi is a general phenomenon in dune soils. The actual increase of the CPs is probably even higher than that indicated by the plate counts. Microscopic observations showed that many bacteria were attached to hyphae, and dispersion of such cells for plate counts may not have been complete.

Unlike those of the CPs, numbers of nonchitinolytic bacteria did not increase in the mycelial zone. This indicates that the amount of hypha-derived materials supporting bacterial growth was small. Therefore, the proliferation of the CPs was probably due to direct mycolytic interaction with the living fungal hyphae. Furthermore, this finding suggests that chitinase was required for growth on living hyphae. The role of chitinase, however, is not completely clear. Proliferation of the CPs in the mycelial zone was inhibited only partly (for Chaetomium and Fusarium) or not at all (for Mucor) by the chitinase inhibitor allosamidin. Additional experiments showed that this was not attributed to ineffectiveness of allosamidin in the soil-like system. Apparently, other factors, in addition to chitinase, contributed to the growth response of the CPs. Bacterial chitinase production has previously been observed in combination with other antifungal factors, such as other lytic enzymes and antibiotics, which may also have been involved in the observed growth response (4, 9).

The ineffectiveness of allosamidin in reducing growth of the CPs in the mycelial zone of Mucor suggests that chitinase is not required by the bacteria in the acquisition of nutrients from this fungus. Unlike the other fungi tested, Mucor incorporates chitosan rather than chitin as a major component of the cell wall (3), and it has been shown that chitosanases are required for protoplast formation in Mucor species (20). The possession of both chitinase and chitosanase is not uncommon for soil bacteria (11). Therefore, chitosanase may well be involved in the response of the CPs to hyphae of Mucor.

The impact of chitinolytic bacteria on fungal development is not yet known, as the actual amount of fungal material converted by bacteria was not determined. However, only partial degradation of the fungal mycelium was observed, as mycelial networks remained visible throughout the incubation period. The growth response of the CPs appeared to be restricted to young hyphae, as their numbers did not increase in the mycelial zones containing only old (>4 weeks) hyphae, suggesting that degradation of mature or empty fungal hyphae in soils is not a niche for them.

The growth response of CPs to invading fungal hyphae supports our hypothesis that nonfilamentous soil bacteria with low chitin-degrading ability use their chitinase genes in interactions with fungi rather than for degradation of chitinous material. This hypothesis, however, should also apply to chitinolytic stenotrophomonads. Yet growth of these bacteria was not much stimulated by fungi. Unlike the CPs, stenotrophomonads isolated from dune soils were not able to degrade colloidal chitin in minimal media but required additional growth factors which could be supplied by adding yeast extract (De Boer and Klein Gunnewiek, unpublished results). Perhaps the lack of these growth factors in the sand suppressed their proliferation in the mycelial zone.

Chitinolytic Cytophaga-like bacteria also showed little response to the presence of living fungal hyphae. In this case, limitation due to lack of additional growth factors is probably not the cause of this poor stimulation, as these bacteria are strong degraders of both colloidal and particulate chitin in minimal media and soil microcosms (11; De Boer and Klein Gunnewiek, unpublished results). Chitinolytic Cytophaga-like bacteria may, therefore, be better equipped to degrade dead chitinous materials than to attack living fungal hyphae.

In conclusion, we present strong evidence for mycolytic growth of chitinolytic -subclass Proteobacteria, which are dominant among the culturable nonfilamentous chitinolytic bacteria in Dutch coastal dune soils. Future research will be directed to the mechanisms and regulation of this type of growth and to the fitness consequences for both bacteria and fungi.