Effect of d-factors on in vitro cerato-ulmin production by the Dutch elm disease pathogen Ophiostoma novo-ulmi

Myca!. Res. 99 (10); 1211-1217 (1995)

1211

['ri"Ied ill Creal Britain

Effect of d-factors on in vitro cerato-ulmin production by the Dutch elm disease pathogen Ophiostoma novo-ulmi

M. LOUISE SUTHERLAND AND C. M. BRASIER Forest Research Station, Alice Holt Lodge, Farnham, Surrey GUlO 4LH,

u.K.

When healthy EAN or NAN race isolates of the Dutch elm disease pathogen Ophiosfoma novo-ulmi were infected with cytoplasmic, virus-like d-factors, they showed reduced produdion in vitro of the protein wilt toxin cerato-ulmin. Isolates infected with the d 3 , d 7 , d9 and d ll factors showed significant decreases in cerato-ulmin production (P < 0'05), producing little detectable toxin. D-fadors d 1 , d 4, dO, d 6 and dB caused moderate reductions (P < 0'05), but the levels were within the lower ranges previously recorded for healthy EAN and NAN isolates in shake culture. When different NAN isolates of the same vegetative compatibility type (the European NAN v-c supergroup) were each infected with the d 2 factor, the reductions in cerato-ulmin production varied with the background genotype. When EAN isolate Pl14 was infected with a range of different d-factors, cerato-ulmin production was found to be strongly positively correlated with both radial growth rate and final in vitro biomass, and thereby negatively correlated with the severity of the d-factors involved. The significance of these results is discussed in terms of the influence of d-factors on hyphal growth and development.

The first and second pandemics of Dutch elm disease from ca 1920-40 and 1940 to the present day have been caused by the pathogens Ophiostoma ulmi (Buisman) Nannf. and 0. novoH/mi Brasier respedively (Brasier, 1991). The more highly aggressive O. novo-H/mi, responsible for the current pandemic, exists as two biotypes, the North American (NAN) and the Eurasian (EAN) races, which are partly reprodudively isolated, differ in a range of morphologicaL physiological and molecular charaderistics and have different geographical distributions (Brasier, 1979, 1986 a, 1990 a, 1991; Jeng, Bernier & Brasier, 1988; Brasier et al., 1993; Bates, Buck & Brasier, 1993 a, b). Within 0. novo-ulmi populations, cytoplasmically transmitted virus-like agents termed d-fadors occur which can exert a deleterious effed on the development and pathogenicity of the fungus (Brasier, 1983, 1986b; Webber, 1987, 1993), and can strongly influence its survival and population structure (Brasier, 1988, 1990 b). Many different d-factors have been identified, some of which have been numbered sequentially from d 1 to d n (Brasier, 1986b; Rogers, Buck & Brasier, 1988). In vitro, d-infeded isolates are characterized in particular by reduced growth rates, unstable ' amoeboid' colony morphologies, low spore viability and decreased reproductive vigour (Brasier, 1983, 1986b). Under field conditions, Webber (1987,1993) showed that d-fadors d 2 , d 6 and d 13 significantly increased the number of spores required for infedion of English elm, Ulmus procem Salisb., via artificial beetle feeding grooves. D-factors therefore have the potential to break the cycle of Dutch elm disease (Webber, 1987, 1993). Most d-fadors studied are associated with multiple doublestranded RNA (dsRNA) segments (Rogers, Buck & Brasier, 1986, 1988; Rogers, 1987). Some dsRNA profiles result in abnormal mitochondrial fundion and the indudion of

mitochondrial plasmids (Rogers, Buck & Brasier, 1987; Charter, Brasier & Buck, 1993a; Charter, Buck & Brasier, 1993 b). 0fadors also share certain similarities, including dsRNA, with some forms of transmissible hypovirulence in the chestnut blight fungus Cryphoneetria parasitica (Murrill) Barr (Day et aI., 1977; Van Alfen, 1986). Transmissible hypovirulence fadors are associated with a natural remission of chestnut blight in parts of Europe (e.g. Grente & Saure!, 1969; Griffin, 1986). It has recently been reported that in Cryphonedria parasitica certain hypovirulence dsRNAs caused dired down regulation of the products of a number of sporulation and virulence genes, including produdion of the polypeptide cryparin (Carpenter et al., 1992; Villalon, Zhang & Van Alfen, 1992). Cryparin is a cell surface protein which has similar physical properties and external accumulation sites to cerato-ulmin (Carpenter et a/., 1992), a low molecular weight protein produced in considerable quantity by 0. novo-ulmi in liqUid shake cultures and on the surfaces of its hyphae and synnemata (Takai, 1974, 1980; Takai, Richards & Stevenson, 1983; Nordin et al., 1987; Richards & Takai, 1988; Svircev, Jeng & Hubbes, 1988; Brasier et al., 1990; Richards, 1993; Yaguchi et al., 1993). Cerato-ulmin is believed to function as a cell-surface hydrophobin (Stringer & Timberlake, 1993; Brasier, Kirk & Tegli, 1994). In addition, it is considered to be a toxin involved in the development of Dutch elm disease symptoms in trees infected with O. ,!Ova-II/mi. It can be detected immunologically in diseased elms, and at low concentrations induces symptoms similar to those of natural infection including reduced water uptake, wilting, chlorosis and necrosis of leaves and browning of the vascular tissue in healthy Ulmus americana L. cuttings (Takai, 1974, 1980; Takai et aI., 1983; Nordin et al., 1987; Richards & Takai, 1988;

Effect of d-factors on CD production

1212

Svircev et al., 1988; Richards, 1993). The toxicity of ceratoulmin also shows some host specificity to elm (Takai et al., 1979). Furthermore, Takai (1974, 1980) and Brasier et al. (1990) showed major differences in the in vitro production levels of cerato-ulmin between the two Dutch elm disease fungi, O. ulmi and 0. novo-ulmi; the production indices for the former ranging from only 0 to 25 compared with 50-850 for EAN and NAN isolates. The higher levels of cerato-ulmin production by 0. novo-ulmi are considered an important factor in its higher levels of pathogenicity to elm (Brasier et al., 1990). In view of the potential of d-factors for the control of Dutch elm disease and the observation of down regulation of cryparin production by dsRNA in C. parasitica, an investigation of the effect of d-(actors on cerato-ulmin production by O. novo-ulmi was of considerable interest.

MA TERIALS AND METHODS Source of isolates and culture methods The geographical sources and EAN or NAN race affiliation of the 0. novo-ulmi isolates used are shown in Table 1. Isolate Logl/3-8d 2 tol (hereafter called Ld) is a derivative of isolate W2tol which became naturally d-infected in a field experiment. W2tol was inoculated into the xylem of a standing tree and acquired the d 2 factor from wild NAN isolates brought into the bark of the tree by breeding elm bark beetles (see Webber & Brasier, 1984). Experiments were constructed according to the availability of different d-factors within the various vegetative compatibility (v-c) groups. Stock cultures were maintained on 2 % Oxoid malt extract agar (MEA; Brasier, 1981) plates at 20-22 DC in the dark and

subcultured at 3 wk intervals. Long-term stock cultures were stored both on 2 % MEA slopes at - 20 0 and under liquid nitrogen. Isolates for dsRNA extraction were grown on 20 ml of 2 % MEA in 9 cm Petri dishes overlain with deplastized cellophane discs (Rogers et al., 1986; Rogers, 1987) and maintained in the dark for 14 d at 20 0 . Growth rate and colony morphology comparisons were used to assess isolates for the presence of d-infection. Mean radial growth rates per day were calculated from colony diameters measured after 2 and 7 d growth on 2 % MEA at 20 0 in the dark (Brasier, 1981). Their colony morphologies were assessed after a further 10 d in diffuse light (Brasier, 1981).

V-factor transfer D-faetors can be freely transferred from diseased to healthy isolates of the same v-c genotype via a 'd-reaction' (Brasier, 1983, 1984, 1986b). A single locus nuclear gene marker for MBC (benomyl) tolerance (Brasier & Gibbs, 1975; Webber, 1988) was used to confirm that a healthy MBC tolerant recipient isolate had been transformed by a d-infected donor (Brasier, 1983, 1986 b). Healthy and d-infected isolates were paired 1 cm apart on each of four replicate elm sap wood agar (ESA) plates (Brasier, 1981). Cultures were grown at 20 0 in the dark for 9 d and then transferred to diffuse light for 10 d or until each colony had reached the edge of the Petri dish. Subcultures from resulting d-reaction zones (see Brasier, 1983, 1986 b) were assessed for colony stability on 2 % MEA (Brasier, 1986 b), for sensitivity to benomyl MBC fungicide (Brasier & Gibbs, 1975), and for transfer of dsRNA (see below).

Table 1. Geographical origins and vegetative compatibility types of Ophiostoma ulmi and O. novo-ulmi isolates Isolate no.

v-c group

Source

Country

Location

Sampled by

Date

0. novo-ulmi NAN HI06 H301 H321 H363 H623 H625 Bl/3d B69/7 BP6-1-10 CI03 CI13 Log 1/3-8d 2 tol (Ld) W2 W4

NAN supergroup NAN supergroup NAN v-c group 2 NAN supergroup NAN supergroup NAN supergroup NAN v-c group 4 NAN v-c group 4 NAN v-c group 4 NAN v-c group 3 NAN v-c group 3 NAN supergroup NAN supergroup NAN supergroup

Twig Twig Twig Twig Twig Twig Beetle Beetle Ascospore Twig Twig MBC tolerant Twig Twig

France Ireland France Ireland

J. Pinon

Spain Spain

Paris Castlenock Bordeaux Caledon Jersey Jersey Guadalajara Guadalajara

UK UK

Chichester Chichester

1977 1978 1979 1979 1981 1981 1984 1984 1984 1982 1982

UK UK

0. novo-ulmi EAN H254 P99B P104 P109 Pll1 P114 P123 RI08 R114

EAN EAN EAN EAN EAN EAN EAN EAN EAN

Bark Twig Twig Twig Twig Twig Twig Twig Twig Twig

0. ulmi R21

supergroup supergroup supergroup supergroup supergroup supergroup supergroup supergroup supergroup

Tewkesbury Kent

A. Mangan H. Heybroek D. Seaby N. J. Bradshaw N. J. Bradshaw J. F. Webber J. F. Webber J. F. Webber C. M. Brasier C. M. Brasier J. F. Webber J. N. Gibbs J. N. Gibbs

1972 1972

Russia Poland Poland Poland Poland Poland Poland Romania Romania

Moscow Kostrzyn Sarbinovo N. Sarbinovo S. Chojna Troszyn Swinoujscie Dorohoi Dorohoi

H. Heybroek C. M. Brasier C. M. Brasier C. M. Brasier C. M. Brasier C. M. Brasier C. M. Brasier C. M. Brasier C. M. Brasier

1978 1980 1980 1980 1980 1980 1980 1980 1980

Romania

Bozovia

C. M. Brasier

1980

UK UK

1213

M. Louise Sutherland and C. M. Brasier

Extraction and analysis of dsRNA All isolates were analysed for the presence of dsRNA. Mycelium from 14-d-old cultures grown on cellophane discs was dried on a Buchner funnel using a Javac DL75 pump for 30 min and finely ground in liquid nitrogen. Double-stranded RNA was extracted according to the technique of Morris & Dodds (1979) as modified by Rogers et al. (1986). One-dimensional PAGE was conducted as described by Rogers et aI. (1986) using 1'5 mm thick vertical slab gels with a 160 mm 4 % acrylamide gel prepared from a stock solution of 40% (w/v) acrylamide and 0'4% (w/v) N, N'-bismethylene acrylamide. Electrophoresis was carried out in TAE buffer (40 mM Tris, 1 mM EDTA, adjusted to pH 8'2 with acetic acid) at 30 V (constant voltage) for 16-18 h (Jamil, Buck & Carlile, 1984). Double-stranded RNA samples were suspended in TE buffer (100 mM Tris, 0'2 mM EDTA, pH 7'5) with 10% RX sample buffer (lOX TAE buffer, 50% glycerol, 0-5 M EDTA, 0'1% SDS and 0-03% bromophenol blue). Multiple virus-like dsRNA segments from Penicillium stoioniferum Thorn (Bozarth, Wood & Mandelbrot, 1971) and Aspergillus foetidus (Nakaz.) Thorn & Raper (Ratti & Buck 1972; Buck & Ratti, 1975, 1977) were included in the gel as molecular weight standards. Gels were stained with ethidium bromide (1 I-lg ml- 1 ) for 20 min, examined for fluorescent bands under uv light and immediately photographed. Bands were confirmed as dsRNA by their resistance to DNase I and single strand specific Sl nuclease and by sensitivity to RNase A in low ionic strength buffer, as described by Rogers et al. (1986). DNase I, 10 I-lg ml- 1 (Sigma Chemical Co.) digestions were conducted in 0'03 M MgCl 2 at 30° for 20 min (Morris & Dodds, 1979). Digestion with Sl nuclease, 100 units ml-\ was carried out in 0'1 M sodium acetate buffer with 0'05 M MgS0 4 and 5 mM ZnCl 2, pH 4'6, at 37° for 50 min. RNase incubations were conducted in 0'05 % TAE buffer at 37° for 2 h with an enzyme concentration of 250 I-lg ml- 1 .

Assessment of cerato-ulmin production in vitro For each isolate, 3 x 100 ml conical flasks containing 25 ml liquid medium of Takai & Richards (1978) supplemented with 1-2% sucrose (Brasier et al., 1990) were inoculated with 2 x 2 mm plugs from the edge of 7 d MEA cultures. Flasks were incubated on a gyratory shaker at 120 rev. min- 1 at 20-22° for 7 d or as still cultures at 20-22°. Cultures were centrifuged at 1000 g for 20 min to pellet the mycelium and conidia. The dry weight of each culture was determined. The supernatant was filter sterilized through a 0'45 I-lm Millipore membrane and the cerato-ulmin concentration determined by turbidimetry. The cerato-ulmin production index (CPI) was calculated as turbidity = (A 4oo ) x 100 x dilution factor of the sample (Takai, 1978; Takai & Richards, 1978). The ceratoulmin production level of each isolate was determined in two separate experiments.

RESULTS Effects of the d 2 factor So far the d 2 factor is the most intensively studied of d-factors and is well characterized both biologically and molecularly

(Brasier, 1983, 1986 b; Rogers et aI., 1986, 1988; Rogers 1987; Webber, 1987, 1993; Charter et aI., 1993 a, b). It was originally discovered in isolate Logl/3-8d 2 tol (Ld), a natural derivative of the NAN isolate W2tol (Webber & Brasier, 1984). It belongs to the European NAN v-c supergoup (Brasier, 1988). To investigate the effects of the d 2 factor on cerato-ulmin production in a range of genotypes, seven healthy isolates of the European NAN v-c supergroup were newly infected with the d 2 factor via d-reactions and assessed for d-infection. Isolates infected by the d 2 factor are usually characterized by reduced growth rates (Brasier, 1983, 1986b; Webber, 1987, 1993). The growth rates of the newly d 2 infected isolates were therefore analysed. As expected, they showed considerably slower growth than their healthy counterparts (Table 2). All growth reductions were significant (P < 0'05). However, the reductions were not uniform, varying from ca 31 % for isolate W2d 2 to ca 74% for isolate H625d 2 . In all cases they were associated with the development of an unstable amoeboid colony morphology. All healthy isolates and their newly d 2 -infected counterparts were tested for dsRNA (d. Rogers et al., 1988 for dsRNA profiles of some of these isolates). Healthy isolates H301 and H363 did not contain any dsRNA; W2 exhibited two bands of 2'40 and 0'61 x 10 6 daltons, including one doublet; H106 contained two bands of 1'88 and 1'57 x 106 daltons; W4 exhibited three bands of molecular weights 1'75, I-52 and 0'33 x 10 6 daltons; isolates H623 and H625 contained four identical sized bands of molecular weights 2'08, 1'67, I-52 and 0'70 x 10 6 daltons. All the newly d 2 -infected isolates contained the 10 dsRNA segments of the donor isolate Ld, i.e. bands of Table 2. Comparative growth rates of healthy and d-infected NAN and EAN isolates Growth reduction Mean radial growth rate (mm d- l ) '

(%)

Isolate no, healthy j d-infected

Healthy

d 2 -infected

d 2 -infected v. healthy

Test I H321jH321d ' C113jC113d 4

4'4±0'1 4'O±O'I

4'3±0'0 4'5±O'O

+13

Test II HI06/H106d 2 H301jH301d 2 H363 jH363d 2 H623/H623d2 H625 jH625d" W2/W2d 2 W4jW4d'

3'7±0'I 3'7±O'1 3'8±0'0 3'8±0'0 3'8±O'I 3'9±0'0 3'6±O'O

1'5±0'8 2'6±0'2 1'7±O'3 I'2±O'3 1'0±0'2 2'7±O'I 1'8±0'8

59 30 55 68 74 31 50

Test III P114/P114d' P114/P114d 5 P114jP114d 6 P114jPII4d' PII4jPI14d' PI14/PI14d 9 P114/PI14d 'O P114/P114d" BP6-1-lOjBP6-I-lOd '2 BP6-1-10 jBP6-1-lOd13

2'8±0'1 2'8±0'1 2'8±0'I 2'8±O'I 2'8±O'j 2'8±0'j 2'8±0'I 2'8±0'1 3'6±O'O 3'6±0'0

I'I±0'2 2'I±O'8 2'I ±0'2 1'2±O'3 I'9± ]'0 0'6±O'2 2'3 ±O'3 O'9±O'4 O'5±O'1 2'0±O'4

61 25 25 57 32 79 18 68 86 44

• Values represent the mean of 2 replicates ±

S.E.M.

2

Effect of d-factors on CU production

1214

1800

200 180 160 140 120 ;:;: 100 U 80 60 40 20 0

1500 1200 OJ)

;:;: 900

u

300 ~

0

Still Culture conditions

1800

2

1500

,.

1200

:: 900 0..

U

600

600 300

o

L--'----_---l.~:..LL.::..L

Shake

Still Culture conditions

Figs 1-2. Comparison of cerato-ulmin production by healthy (D) and d 2 -infected (0) H363 isolates in 7 d shake cultures and 7 d still cultures. Fig. 1. Cerato-ulmin production. Fig. 2. Cerato-ulmin production per gram final biomass. Vertical error bars represent S.E.M. molecular weights 2'40, 2'08, 1'84, 1'67, 1'60, 1'52, 0'65, 0'61, 0'33 and 0'23 x 10 6 (Rogers et al.. 1986), in addition to any dsRNA bands already present in the recipient. Cerato-ulmin production is usually determined after 7 d in shake culture in which the fungus grows as a budding yeast phase. However, Brasier (1986 b) and Rogers (1987) showed that for d-infected isolates there was a progressive loss of dfactors with each cell cycle in the yeast growth phase, and presumably of the associated dsRNA segments (d. Rogers et al., 1986). Therefore in a preliminary experiment, the effects of d 2 infection on cerato-ulmin production in 7-d-old shake cultures and 7-d-old still cultures were compared, using healthy isolate H363 and its d 2 -infected counterpart H363d 2 . H363 produced the highest level of cerato-ulmin (Cpr = 186) after 7 d in shake culture (Fig. I). However, biomass production varied so greatly between the two growth regimes that final cpr values alone give no indication of the amount of ceratoulmin produced per gram biomass. Analysis of values of cpr g -1 indicated that healthy H363 produced more ceratoulmin per gram final biomass in still as opposed to shake cultures (Fig. 2), and that the decrease in cerato-ulmin production in H363d 2 was most marked in still cultures (Fig. 2). Cerato-ulmin production by the seven healthy isolates and their d 2 -infected counterparts was therefore compared in 7-dold still cultures, and was expressed per gram final biomass to allow for differences in the growth rate of healthy versus d 2 _ infected isolates. Cerato-ulmin levels for the healthy isolates H106, H30I, H363, H623, H625, W2 and W4 were within the normal NAN range in shake culture (Brasier et aI., 1990) (Fig.

W2

~ W4 HI06 H301 H363 H623 H625 O. ulmi control Isolate number

Fig. 3. Comparison of cerato-ulmin production by healthy (D) and d 2 -infeeted (0) NAN isolates of O. novo-ulmi in 7 d still cultures. Vertical error bars represent S.E.M. 3). When d 2 -infected, six out of the seven isolates produced significantly less (P < 0'05) cerato-ulmin; isolate H301d 2 did not produce any detectable amounts of cerato-ulmin; H623d 2 produced very low levels, equivalent to that produced by the control O. ulmi isolate R21. Production by isolates W2d 2 , H363d 2 , W4d 2 and H625d 2 was reduced by ca 75, 64, 53 and 42 % respectively; however, these levels were still within the range for healthy NAN isolates in shake culture (Brasier et al.. 1990). The d 2 factor decreased cerato-ulmin production by isolate H106 only slightly and the reduction was not significant (Fig. 3). Effects of other d-factoys

Twelve other O. novo-ulmi d-factors were selected for a similar study. These comprised four NAN d-fadors (dt, d 4 , d 12 and d 13 ) (Table 1), of which only d 12 and d 13 occurred in isolates of the same v-c group, the Guadalajara v-c group (Brasier, 1988; Webber, 1993); and eight EAN race d-fadors (d 3 , d 5 , d 6 , d 7 , d S , d 9 , d 10 and d Il ) (Table I) all of which were originally identified in isolates of the EAN European v-c supergroup (Brasier, 1988). The dsRNA profiles of all these d-fadors (Rogers, et al.. 1988) and the effeds on the growth and development of dt, d 5 , d 9 and d 13 (Brasier 1983, 1986b; Webber, 1993) have been examined previously. The d 12 and d 13 fadors differ only by a single dsRNA band (Rogers, et al., 1988) and are considered to be minor variants of the same dfador (J. F. Webber, pers. comm.). Each d-fador was transmitted to a healthy isolate of the same race and v-c type via a d-readion. Of the NAN dfadors, d 1 was transferred to isolate H321, d 4 to isolate Cl13, and d-fadors d 12 and d 13 to isolate BP6-1-1O. EAN d-factors d 3, d 5, d 6 , d 7, d S, d 9 , d 10 and d ll were all transferred to the healthy EAN v-c supergroup isolate P1l4. The dsRNA profiles (i.e. the number and molecular weights of dsRNA segments) of the newly derived d-infeded isolates were assessed and compared with that of the donor. The healthy NAN recipient H321 contained two bands of 1'48 and 0'11 x 10 6 daltons, Cl13 contained two bands of 0'31 and 0'13 x 10 6 daltons and BP6-1-10 contained one band of 0'14 x 10 6 daltons. The healthy EAN recipient P1l4 contained no dsRNA segments.

1215

M. Louise Sutherland and C. M. Brasier 2000,-------------------,

2500 r - - - - - - - - - - - - - - - - - - - - - ,

1600

2000

,. 1200

'ell

0:

0:

ell

u 800

1500

U 1000

400

500

O'--'-----'---'----'--'-'-'=---'--J..==-'--~----'

N

M

'" N

M

""0 M

M

:I: :I:

~

~

0

0

~

~

'i':C5

C!l C!l C!l

08

0

U U

~

"0 "0

Fig. 4. Comparison of cerato-ulmin production by healthy (D) and d-infected (E2l) NAN isolates in 7 d still cultures. Vertical error bars represent S.E.M.

NAN d-factors. Of the NAN d-factors, d 12 and d 13 significantly (P < 0'05) reduced the growth rate of isolate BP6-1-1O and induced unstable amoeboid colony morphologies (Table 2). As previously observed by Webber (1993), d 12 was consistently the more debilitating d-factor over three experiments, causing growth rate reductions of ea 69-86 % compared with ea 14-44 % for d 13 . 0 4 caused a slight but not significant increase in growth rate (Table 2) and had no evident effect on colony morphology. Analysis of cerato-ulmin production after 7 d in unshaken liquid cultures showed that d 1 and d 4 significantly reduced cerato-ulmin production per gram final biomass (ea 33-48 % for d 1 and ea 66-90 % for d 4 ; P > 0'05) (Fig. 4). 0 12 reduced cerato-ulmin production slightly and the d 13 factor induced a small increase in cerato-ulmin production but neither change was significant. EAN d-factors. The eight EAN d-factors were initially compared in terms of their effects on growth rate and colony morphology of the recipient isolate P114. All the d-factors decreased the growth rate of P114, but the reductions varied between the different d-factors (Table 2). Moderate growth reductions of ea 18-32 % were caused by d S , d 6 , d 8 and d lO , and greater reductions (P < 0'05) of ea 57-79% by d 3 , d 7 , d 9 and dU. The magnitude of these reductions remained constant over three separate growth rate experiments. All the newly dinfected Pl14 colonies developed unstable amoeboid colony morphologies. Healthy isolate Pl14 produced cerato-ulmin within the normal range for EAN isolates in shake culture (Brasier et al.. 1990). D-factors d 3 , d S, d 6 , d 7, d 8 , d 9 and d U all Significantly (P < 0'05) reduced cerato-ulmin production with respect to Pl14 (Fig. 5), the reductions ranging from ea 38 % with d S to ca 93 % with d 3 . The very low levels of cerato-ulmin produced by P114 when infected with d 3 , d 7 , d 9 and d ll were equivalent to that produced by control O. ulmi isolate R21 (13 CPI g-l), and within the normal range for O. ulmi (Brasier et al., 1990). Although d-factors d S, d 6 and d 8 significantly reduced ceratoulmin production, the levels were still within the normal range for healthy EAN isolates in shake cultures (Brasier et al.,

'""0

ll..

ll..

"0

'=0

~

'""0

ll..

ll..

ll..

ll..

~

'i:C5

"'::s-....c

"" "" "" "" "" "" ""

..a ..a ..a "'::s-....c ll.. ll.. ll..

Isolate number

':0

ll..

ll..

ll..

08

Isolate number Fig. 5. Cerato-ulmin production in 7 d still cultures by healthy isolate PII4 (D) and its counterparts infected with EAN d-factors d 3 , d S , d 6 , d 7, dB, d 9• dID and d ll (E2l). Vertical error bars represent S.E.M. 250r--------------,----, 6 200 ISO

0:

u

•

100 50

~O

60 80 100 Biomass production (mg final D.W.)

120

2500 7

2000 ,

ell

•

1500

c: U 1000

•

500 0 0·0

•

•

•

0·5 \·0 1·5 2·0 2·5 Mean radial growth rate (mm d-')

3·0

Figs 6-7. Relationship between cerato-ulmin production by P114 isolates infected with EAN d-factors. Fig. 6. Biomass production (mg final D.W.) Fig. 7. Mean radial growth rate (mm d- I).

1990). 0 10 had a stimulatory effect, increasing cerato-ulmin production by ca 11 %.

Correlations between growth rate, biomass and ceratou1min production in vitro For EAN d-factors cerato-ulmin production (epI) was positively correlated (r2 = 0'77, D.F. = 7) to biomass production (mg final D.W.) in still culture (Fig. 6). To verify this relationship the experiment was repeated and the correlation between cerato-ulmin and biomass production for the EAN dfactors was r = 0'69, D.F. = 7. With the EAN d-factors a positive correlation (r2 = 0'83, D.F. = 7) was also found between the mean radial growth rate per day and cerato-ulmin production (per gram final biomass) (Fig. 7). O-infected isolates with growth rates less than 1'9 mm d- 1 produced

1216

Effect of d-factors on CU production little or no cerato-ulmin. With d-infected isolates with growth rates above 1'9 mm d- I (Le. d 5 , d 6 , d S and d IO ), the reductions in cerato-ulmin production were directly proportional to growth rate. No such correlations were found for the NAN dfactors examined.

DISCUSSION These results show that, in addition to having a deleterious effect on the growth and colony stability in vitro, d-fadors can significantly reduce the production by 0. novo-ulmi isolates of the protein wilt toxin and hydrophobin cerato-ulmin. The reductions in cerato-ulmin production varied with the d-factor involved, the genotype of the recipient isolate, and with cultural conditions. The relative effects of d-infection on growth rate and cerato-ulmin production by a particular dfactor/recipient combination also varied between experiments. This is to be expected for interactions between processes which, individually, already show complex environmental effects (Brasier, 1986 a; Brasier et al., 1990; Tegli et a/., 1994). Despite this background variability some important trends were detected. Preliminary studies with isolate H363 and the d 2 factor demonstrated that the reduction in cerato-ulmin production was greatest in still culture, in which the fungus grows as a mycelium, as compared to shake cultures, in which the yeast phase predominates. This is likely to reflect the previously reported loss of the d 2 factor and its associated dsRNA segments in the yeast phase and the emergence of a high proportion of healthy bud cells (Brasier, 1986a; Rogers et al., 1986). The main comparisons were therefore conducted with still cultures, which may also parallel the behaviour of dinfected isolates in solid media and in elm bark more closely. The reductions in cerato-ulmin production varied considerably with different d-factors. However, the relative order of reductions caused by different d-factors was consistent over different experiments. D-factors d 3 , d 7, d 9 and d ll reduced the cerato-ulmin production of their healthy counterparts by > 59%, down to levels comparable to those of O. ulmi isolates under the same conditions (Fig. 5) or in shake cultures (Brasier et al., 1990). Seven other d-factors reduced ceratoulmin production moderately, but the levels remained within the published production ranges for EAN and NAN isolates in shake culture (Brasier et al., 1990). Two d-factors, d iO and d I3 , caused no significant reduction in the cerato-ulmin production of their recipients and may even have had a slight stimulatory effect. Transmission of the d 2 factor, the most widely studied of all d-factors, to a range of NAN European v-c supergroup isolates showed that reductions in cerato-ulmin production were also influenced by the background genotype of the recipient isolate. The d 2 factor is known to alter the mitochondrial function of the recipient, interfering with cytochrome oxidase activity and inducing the de novo production of mtDNA plasmids (Rogers et al., 1987; Charter et al., 1993 a, b). This variation may therefore reflect differences between the cytoplasmic genotypes of the recipients rather than their nuclear genetic backgrounds. A clue to the nature of the interaction between d-infection and cerato-ulmin production was provided by the response of EAN isolate PII4 to infection by eight different d-factors.

These observations showed that cerato-ulmin production by still cultures was positively correlated both with their final biomass and with the linear growth rates of the different PII4/d-factor combinations on 2 % MEA. For linear growth rate there was a critical threshold of ca 1'9 mm d- I below which little or no cerato-ulmin production was detected for that combination in the liquid cultures. Above this threshold the quantity of cerato-ulmin produced was strongly proportional to growth rate. Wessels (1992), Wosten, Devries & Wessels (1993) and Wosten et al. (1994) showed that in Schizophyllum commune Fr. hydrophobins affect hyphal development and are secreted into the medium only by actively growing hyphae. Recently cerato-ulmin has been identified as a hydrophobin based on its hydrophobicity plot (Stringer & Timberlake, 1993) and on evidence of its effect on mycelial development (Brasier et al., 1994). The debilitating effects of d-factors on O. novo-ulmi colonies may result from a form of premature hyphal senescence (d. Brasier, 1986a; Charter, et al., 1993a, b), with consequent decreases in hyphal extension and biomass. The above correlations therefore suggest that d-infedion may indirectly reduce the amount of cerato-ulmin excreted into the medium via its effect on inhibition of growth, rather than directly affecting the cellular production of cerato-ulmin as has been suggested for the reduction of cryparin production in dsRNA infeded isolates of C. parasitica (Carpenter et al., 1992; Villalon et al., 1992). We would like to thank the Pilkington Charitable Trust for funding this research.

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