Mycol. Res. 100 (12): 1428-1430 (1996) Printed in Great Britain 1428

Carbon allocation patterns in fungi in the presence of chitin in the external medium

ANGELA HODGElvZ*, IAN J. ALEXANDER1, GRAHAM W. G O O D A Y Z A N D KEN KILLHAM1 ' Department of Plant and Soil Science and Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen AB9 2UE, U.K.

A number of fungi were grown on defined media containing uniformly labelled [14C]glucose with and without additional chitin, to examine how the presence of chitin affected growth and the distribution of 14C between cytoplasm and cell wall. Chitin significantly enhanced the growth of Heterobasidion annosum, Boletinus cavipes and Paxillus involufus. These fungi, with the exception of H. annosum, also incorporated a lower proportion of assimilated 14C into their cell walls. The specific activity of 14C in the fungi screened was generally reduced in the presence of chitin indicating incorporation of C from chitin in fungal tissue. In the case of Phytophthora cinnamomi there was a significant increase in specific activity. These data indicate that patterns of carbon allocation in both chitinous and non-chitinous fungi change in the presence of chitin in the external medium.

A marked growth response by some fungi grown in media containing chitin, glucose and (NH4),HP04, compared to when glucose and (NH4),HP04 alone were supplied, has been reported by Hodge, Alexander & Gooday (1995). Mycorrhizal fungi, particularly the ericoid endophyte Hymenoscyphus ericae, can grow on chitin when it is supplied as the sole nitrogen source (Leake & Read, 1990). H. ericae has also been shown to utilize N-acetylglucosamine as a sole N source (Bajwa & Read, 1986). However, chitinolytic activities may be repressed in the presence of glucose (Blaiseau et al., 1992; Ulhoa & Peberdy, 1993) and the relationship between extracellular chitinolytic activities and fungal growth in pure culture is poor (Hodge e f al., 1995). To examine further the mechanism by which chitin stimulates the growth of fungi already supplied with glucose we investigated the fate of carbon from glucose in the presence of exogenous chitin. A number of fungi were grown on 14C-labelled glucose with or without unlabelled chitin. It was hypothesized that incorporation of carbon from chitin would reduce the specific activity of 14C in the fungi and the extent of the dilution in different compartments (cytoplasm and cell wall) would indicate the fate of the C originating from chitin.

METHODS A N D MATERIALS

The fungi studied were Boletinus cavipes (Opat.) Kalchbr., Paxillus involutus (Batsch) Fr., Pisolifhus tinctorius (Pers.) Coker & Couch, Suillus variegafw (Fr.) Kuntze (ectomycorrhizal basidiomycetes); Armillaria ostoyae Romagn., Heterobasidion annosum (Fr.) Karst. (root-infecting basidiomycete pathogens); Phytophfhom cinnamomi Rands (root-infecting oomycete

' Present address: Plants Division, The Macaulay Land Use Research Institute, Aberdeen AB9 2QJ. U.K.

pathogen) and Trichoderma harzianum Rifai (ascomycete). Cultures were maintained on modified Melin-Norkrans (MMN) agar (Marx, 1969) at 20 OC in the dark. For experimentation, 9250 kBq of uniformly labelled ['4C]glucose (D-glucose-UL-'4C) in aqueous solution (Sigma Chemical Co.) was divided into two equal portions and each mixed with non-radioactive glucose to give a final concentration of 10 g I-'. After autoclaving, the glucose solutions were added separately to two flasks of basal MMN solution containing (NH4),HP04 as N source and from which the malt extract had been omitted. One of the flasks also contained purified crystalline chitin (0.5 g I-') (Sigma Chemical Co.). Portions (20 ml) of the two media were dispensed separately into a series of sterile Petri dishes (90 mm x 15 mm). Thus each Petri dish contained 92.5 KBq of ['4C]glucose. Fungal discs (5 mm) were cut from the edge of actively growing colonies of the test fungi and left to regenerate on Cellophane on half strength MMN agar for 5 d, before transfer to the liquid media (Leake & Read, 1990). One fungal disc was carefully floated on the surface of each of the culture solutions and the dishes kept in the dark at 20° for 22 d. There were six replicate Petri dishes for each fungus and each treatment. At the end of the incubation period the culture solution in the Petri dishes contained in excess of 60 kBq of 14C. While some of this activity may have come from C exuded from hyphae, these levels of activity strongly suggest that excess glucose remained in the culture media of all fungi.

After 22 d, the mycelia from three of the six replicate Petri dishes were gently removed using forceps, washed in distilled water and placed on a previously dried (24 h at 50°) and weighed nylon membrane (3 cm diameter, mesh size 0.45 pm), then oven-dried for 6 h at 50° prior to weighing. The membranes containing the dried and weighed fungi were then cut into sections (5 rnm x 5 mm), placed in a Universal bottle

Angela Hodge and others 1429

Table 1. Uptake of 14C (on a dry weight basis) horn glucose and distribution of 14C in the mycelium of fungi grown in the presence (+chitin) and absence (-chitin) of chitin in the medium (data are means f standard error, n = 3)

Dry Weight (mg)' Specific ActivityZt "C Ratio wall:tota13

-chitin + chitin P value -chitin +chitin P value -chitin +chitin P value

B. cavipes 7.33 k 064 17.94 f 242 0.006 4647 k 203.6 P. invo/utm 7.68f0.50 15.76 k 1.48 0.003 4119k396.6 P. finctoriw 13.61 k 1.52 13.85 k 1.50 0917 4273 k 108.0 5. variegatw 13.34 f 0.83 12.32 f 0.80 0426 4625 f 3475 A. ostoyae 3.75 f 1.47 731 f 0.70 0.117 4452f582.7 H. annosum 8.51 f0.18 27.38 f 2.42 < 0.001 3725 1243.9 T. harzianum 17.51 f0.62 1940 f 0 3 2 0.058 2701 f421.5 P. cinnamomi 9.88f 0.20 929k0.31 0.181 1152f 135.1

Difference due to the presence of chitin tested by t statistics. Data shown t Specific activity units = disintegrations min-I mg-I dry weight.

1077 f 121.5 < 0001 063 k 0.027 039 + 0.056 0014 703 k 73.0 < 0.001 060f 0.025 0.51 t0 .013 0.041

2820+8590 0.103 0-64t0.077 @47+0.012 0.116 1893 f 160.1 0.001 048f0.068 0 5 1 f 0 0 6 4 0812 1936 k 4265 0.026 0.47 f 0023 0 3 1 + 0058 0063 644 k 20.5 < 0901 0 3 1 k 0033 0.42 + 0022 0058

2049 f 1582 0.213 089 + 0.009 0.64 + 0058 0008 1762k33.7 0.018 028 f 0027 0.57 t o 0 4 4 0006

were transformed before statistical analysis by: ' Log,,, 2Squareroot, 3Arcsine.

and digested by the method of Dalal (1979). Prior to digestion, a small vial containing 5 ml 0.4 M NaOH (to trap evolved CO,) was placed inside the Universal bottle which was then stoppered tightly. After digestion was complete, the trap was removed and 3 ml of NaOH were placed in a plastic vial with 2 ml Picofluor 40 scintillation fluid. Radioactivity was measured using a liquid scintillation counter with the inclusion of appropriate controls to account for background radiation. The volume of the NaOH remaining was recorded. This procedure gave the total 14C in the mycelium at 22 d and was used to calculate specific activity.

To determine the proportion of 14C from the labelled glucose in the cytoplasmic and cell wall components, the remaining three replicates for each fungus and each treatment were placed on previously dried and weighed nylon membranes (mesh size 0.45 pm). The fungi and membranes were then each placed on watchglasses (3 cm diam.) and transferred to a desiccator. A beaker containing 100 ml of acid washed chloroform was placed in the centre of the desiccator and the fungi were fumigated with chloroform overnight and then the mycelium and membrane were extracted with 3 x 5 ml 0.5 M K,S04 to remove the cytoplasmic material (Parkinson, Killham & Wainwright, 1990). Three ml of K,S04 solution were removed for scintillation counting. The material remaining on the nylon membrane (i.e. cell wall) was oven dried at 50° for 6 h, weighed and digested (Dalal, 1979) as before. Determination of total carbon in the separate cytoplasmic and cell wall components was not carried out because of the presence of residual chloroform. Total 14C in the mycelium was expressed as the sum of the cytoplasmic and cell wall fractions.

Differences due to the presence of chitin were tested statistically by the t-test (Table I). In order to stabilize the variance between the treatments it was hrst necessary to transform the data. The transformations used were log,, for the dry weight, square root for the specific activities and an arcsine transformation for the 14C wall: total ratio results.

RESULTS A N D DISCUSSION

Chitin significantly enhanced the growth of some fungal isolates. The growth response was greatest for H. annosum, B. capives and P. involuttls (221.7%, 144.7%, 105.2%, respect-

ively). The ascomycete T. harzianum also showed a small (10.8%; P = 0.058) increase in growth. Although growth of A. ostoyae increased markedly (949%) in the presence of chitin, this was only significant at P = 0.117. Growth of P. tinctoriw, S. variegatus and P. cinncrmomi did not alter appreciably (i.e. by f 10%) in the presence of chitin. These results contrast with our previous study, conducted over a 30 d period, where growth of all chitinous fungi screened was significantly increased, and growth of P. cinnamomi decreased, by the addition of chitin to the medium (Hodge et al., 1995).

Specific activity (expressed as disintegrations min-' mg-' D.w.) of all species, except P. cinnamomi, was reduced in the presence of chitin (Table I). This reduction was greatest in P. invofukus (486%), H. annosum (478%), B. cavipes (332%)' S. variegatus (144%) and A. osfoyae (130%). P. finctoriw and T. harzianum showed a smaller and insignificant (P > 0.005) specific activity (52 % and 32 %, respectively). In contrast, P. cinnamomi exhibited an increase (53%: P = 0.018) in specific activity when chitin was present. The ratio of 14C in the wall compared to that in the whole fungus showed that in T. harzianum, B. cavipes and P. involuttls there was a reduction (39% 62% and 18%, respectively) in the proportion of assimilated 14C in the wall when chitin was present. In contrast, P. cinnamomi showed a large (104%: P = 0.006) increase in the proportion of 14C in its wall in the presence of chitin. There was no statistically detectable difference in the wal1:total 14C ratio for the other fungi in the presence or absence of chitin (Table I). The responses of the fungi to chitin are summarized in Figure 1.

All the fungi, except H. annosum, which showed enhanced growth in the presence of chitin also incorporated a lower proportion of assimilated 14C from glucose into their cell wall. This could indicate the incorporation of C from chitin preferentially into the cell walls of these fungi. Alternatively, there may be some reduction in overall carbon allocation to cell walls in the presence of chitin (e.g. thinning of cell walls) thus 'diluting' 14C from glucose. It is unclear how C from chitin becomes integrated into the mycelium of these fungi since extracellular chitinolytic activities under the experimental conditions may be expected to be low, as shown by our previous study (Hodge et al., 1995). One explanation is that the fungi incorporate incompletely hydrolysed chitin frag- ments directly into their walls. However, the a-chitin present

Carbon allocation in the presence of chitin

activity Less specific activity Greater specific activity

+ Chitin

B. cavipes H. annosum i? tinctonus S. variegahcs None None P cinnamomi l? involutus A. ostoyae Z hanianum

Growth stimulated (2 10%)

Fig. I. Diagram illustrating the biological response of the eight different fungal species tested when chitin was present in addition to [14C]glucose and (NH4),HP0,.

No sizeable growth stimulation (< 10%)

in fungal cell walls is a highly ordered crystalline macromole- cule with extensive intra- and inter-chain hydrogen bonding and is covalently crosslinked to glucans. The incorporation of partially hydrolysed chitin fragments into this ordered network seems unlikely on the basis of current knowledge. In the case of H. annosum, a prolific producer of chitinolytic enzymes (Hodge et al., 1995), no significant change in allocation of 14C to walls was observed. Perhaps the chitinolytic system degrades the chitin polymer completely before incorporation occurs and thus C from chitin is incorporated in the same manner as C from glucose. Further experimentation is required before the enhanced growth of some fungi in the presence of chitin when adequately supplied with glucose + (NH,),HPO,, and the fate of the C from chitin, can be explained. Clearly, it is important to ascertain the relative extent to which C from glucose and chitin is released in respiration. The use of 14C- chitin and N-acetylglucosamine would be useful in this respect.

In contrast to the other fungi examined, P. cinnamomi had a greater proportion of assimilated 14C in the cell wall in the presence of chitin, indicating a resource diversion, possibly to increase wall thickness. Increase in cell wall thickness may be a defence response by P. cinnamomi. Suspension-cultured tomato cells have been observed to respond to chitin fragments with a rapid transient alkalinization of the culture medium (Felix, Regenass & Boller, 1993) but the signal transduction pathway is unknown. The chitin used in this study was crystalline a-chitin which is structurally similar to that in fungal cell walls (Aronson, 1965; Bumett, 1979). It would therefore be interesting to investigate whether P. cinnamomi also produces this response in the presence of chitinous fungi.

Although growth of P. cinnamomi was not significantly reduced by chitin, this organism showed markedly different trends in carbon allocation compared to the other fungi screened (Fig. 1; Table I). Specific activity of 14C in the whole mycelium of the chitinous fungi was generally lower when there was chitin in the medium. This could indicate more carbon from glucose was released as CO, or as metabolites

into the media in the presence of chitin. In contrast, specific activity in the mycelium of P. cinnamomi increased. Our data suggest an alteration in carbon allocation patterns within the mycelium of both chitinous and non-chitinous fungi in response to the presence of an external source of chitin.

A.H. was in receipt of a studentship from the Science and Engineering Research Council (now BBSRC), U.K. during the tenure of this work.

REFERENCES

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Bajwa, R. & Read, D. J. (1986). Utilization of mineral and amino N sources by the ericoid mycorrhizal endophyte Hymenoscyphus ericae and by mycorrhizal and non-mycorrhizal seedlings of Vaccinium. Transactions of the British Mycological Society 87, 269-277.

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Bumett, J. H. (1979). Aspects of the structure and growth of hyphal walls. In Fungal Walls and Hyphal Growth (ed. J . H . Bumett & A. P. J. Trinci), pp. 1-25. Cambridge University Press: Cambridge, U.K.

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Marx, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163.

Parkinson, S. M., Killham, K. & Wainwright, M. (1990). Assimilation of 14C0, by Fusarium oqsporum grown under oligotrophic conditions. Mycological Research 94, 959-964.

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(Accepted 17 March 1996)