Molecular cloning of two chitinase genes from Serratia marcescens and their expression in Pseudomonas species

Physiological and Molecular Plant Pathology (1988) 33, 483 491

Molecular cloning of two chitinase genes from Serratia marcescens and their expression in Pseudomonas species )LIF SUNDHEIMt, ALAN R . POPLAWSKY Department

of Plant

and

ALBERT H . ELLINGBOE

Pathology, University of Wisconsin-Madison . Madison, Wisconsin 53706, U .S .A .

(Accepted, for Publication May 1988)

Two chitinase encoding EcoRI fragments from the enteric soil bacterium Serratia marcescens were cloned . From a genomic library of 5686 transductants, 21 expressed chitinase activity as indicated by clearing of a chitin-containing medium. The chitinase encoding clones could be divided into two groups. Four had an 18 kb EcoRI fragment and 17 had a 9-4 kb EcoRl fragment . In Southern hybridization experiments the 18 kb fragment showed no homology to the 9 .4 kb fragment and restriction enzyme maps indicated no similarity . Triparental mating with fluorescent Pseudomonas spp . yielded transconjugants that expressed chitinase activity, inhibited growth of Fusarium o .xysporum f. sp . redolens germ tubes and reduced disease of radish caused by the same fungus .

INTRODUCTION

Chitin is a major component of the cell walls of fungi, except for the class Oomycetes [14] . Insects, nematodes and most other invertebrates have chitin as a structural component of their exoskeletons, while vascular plants and mammals lack chitin [14] . Bacteria [13], fungi [4], animals [14], and higher plants [2, 16] commonly have enzymes (chitinases) that catalyse the hydrolytic degradation of chitin to chitodextrins. The production and secretion of chitinases by nonpathogenic micro-organisms may be important in the biological control of plant pathogenic fungi . The addition of chitin-containing amendments to soil has been observed to reduce plant diseases caused by fungi [12,15,19, 20, 21 ] . The addition of chitin-containing amendments has also been reported to affect nematodes [11] . Serratia marcescens, a Gram negative, enteric soil bacterium, produces and secretes high levels of chitinases . It was the most efficient producer of chitinases among 100 organisms tested [13], and has been the object of investigations into the genetic basis of chitinase production . In one study, a cosmid library of S . marcescens was prepared and used to transform Escherichia coli genetically [6] . Transformants with chitinase activity contained either of two different cosmid clones . The authors concluded that there were two chitinase genes producing two distinct forms of the enzyme . One of the chitinase genes was mutagenized tPresent address : Norwegian Plant Protection Institute, Division of Plant Pathology, 1432 As-NLH, Norway . Abbreviations used in text : CA, chitin agar; NBY, nutrient broth yeast extract ; PD, Potato dextrose ; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside . 0885-5765/88/060483+09 $03 .00/0 © 1988 Academic Press Limited

484

L . Sundheim et al. TABLE I Bacterial strains and plasmids

Strains and plasmids/cosmids Serratia marcescens BJ L200 Escherichia coli HB101 TBI Pseudomonasfluorescens NRRL B-15135 Gram negative Ill Cloning vectors/recombinant plasmids/cosmids pLAFR3 pLESI pLES2 pBR325 pLES3 pLES4

Antibiotic resistance or other traits'

Reference or source

Chi +

Rif, Nal, Ab+

ARS Patent Collection (R. J . Cook)

Rif, Nal, Ab+

[91

Tc, ChiTc, Chi+ (9 .4 kb insert) Tc, Chi+ (18 .0 kb insert) Ap, Cm, Tc, ChiAp, Tc, Chi + (9 .4 kb insert) Ap, Te, Chi + (18 .0 kb insert)

[5] This study This study [11 This study This study

'Rif=rifampicin ; Nal=nalidixic acid ; Tc= tetracycline ; Cm=chloramphenicol ; Ap= ampicillin ; Chi+ =chitinase positive ; Chi- =chitinase negative ; Ab+ =antibiosis positive .

and recombined into the parental S . marcescens genome . This transconjugant showed reduced chitinase and anti-fungal activities . In a separate study, five different chitinolytic proteins with molecular sizes of 21, 36, 48, 52, and 57 kD were identified in S . marcescens, and an EcoRI fragment of 9 .5 kb apparently encoded the 57 kD chitinase [5] . A third study yielded mutants of S . marcescens with two or three times the wild type level of chitinase activity [18] . The objectives of this study were to identify and clone chitinase genes from S . marcescens, transfer these genes to root-colonizing bacteria, and determine their utility in the biological control of plant disease .

MATERIALS AND METHODS Microbial isolates The bacterial strains and plasmids used are listed in Table 1 . Pseudomonasfluorescens strain NRRL B-15135 was obtained from the Northern Regional Research Laboratory in Peoria, IL, where it had been deposited by Dr R . J . Cook, Pullman, WA . Bacterial strain III is a plant growth-promoting rhizobacterium, and was obtained from Dr J . Parke, University of Wisconsin-Madison, WI . Both strains are effective in suppressing take-all of wheat caused by Gaeumannomyces graminis var . tritici (17] . The Fusarium oxysporum f. sp . redolens and F. oxysporum f . sp . conglutinans isolates used in this study were obtained from Dr P . H . Williams, University of Wisconsin-Madison, W1 . The isolate of G . graminis var . tritici used was obtained from diseased wheat in Wisconsin .

Molecular cloning of two chitinase genes

485

Biochemicals and enzymes Restriction endonucleases, T4 ligase and the "Packagene" extracts for in vitro packaging of cosmids were purchased from Promega Biotec, Madison, WI, and used according to the manufacturer's instructions . Bacterial alkaline phosphatase was obtained from International Biotechnologies, New Haven, CT . All antibiotics were from Sigma Chemical Co ., St Louis, MO . 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was purchased from Boehringer Mannheim . (Alpha 32 P) dATP at 600 Ci/mmol was obtained from Amersham . Media E. coli was cultured on Luria-Bertoni (LB) medium [10] . Other bacterial isolates were cultured in nutrient broth yeast extract (NBY) medium [24] or King's B medium [8] . Potato dextrose medium was made from 250 g potatoes, 20 g dextrose, and 1000 ml water . When appropriate, media were supplemented with ampicillin at 50µg ml - ', chloramphenicol at 30µg ml -1 ; kanamycin at 50µg ml -1 ; nalidixic acid at 100 .tg ml -1 ; rifampicin at 100 pg ml - ' ; and tetracycline at 12 .tg ml - ' to produce selective media .

Chitinase assay A solid medium was used to screen clones for chitinase activity . Chitin purchased from Sigma Chemical Co . was purified using the method of Vessey and Pegg [23] and stored as a wet paste at 4 °C . Chitinase assay plates had a base of 15 ml of DM5, which contained 25 mm TES (Sigma) pH 7 . 0, 5 mm (NH4 ) 2 SO4 , 10 mm phosphate buffer (pH 7 . 0), 0 . 1 mm MgSO 4, 5 mm glucose, 0 . 25%2 x TS (2xTS is a solution of 4 g of CaC1 2 .2H 2 0, 2 g of MnS0 4 .H 2O, 1 g of FeS0 4 .7H 2 0, and 0 . 05 g of ZnS0 4 .7H 2O in 1 10* 1 N HCl) and 2° agar . The top 10 ml layer on the double layer chitin agar (CA) plates contained 10° 0 purified chitin and 2°/a agar . Chitinase activity on the plaque medium is visible as a zone of clearing around colonies which produce and secrete chitinase . Molecular techniques Whole cell DNA was isolated from S . marcescens strain BJL 200 by the methods of Keen et al . [7] . Agarose gel electrophoresis, DNA nick translation, Southern hybridization, and genomic clone bank constructions were performed using standard techniques [10] . Construction of genomic library The cosmid vector pLAFR3 [3, 22] was cut with EcoRl and treated with bacterial alkaline phosphatase . Ligation of vector to size fractioned S . marcescens BJL 200 DNA from a partial EcoRI digest was carried out at 14 °C for 24 firs . Recombinant cosmids were packaged in vitro using the "Packagene" extract and transduced into E. coli HB101 . Following overnight growth on LB medium containing Tc, transductants were transferred to master plates and replica plated to chitin agar overlay plates . Biocontrol experiments The bacterial strains were increased for 48 h at 29 ° C on NBY incorporating the appropriate antibiotics, centrifuged, and resuspended in one tenth of the original volume of 1" 0 carboxymethyl cellulose . Radish seed of the cv . Icicle Short Top was coated with

486

L . Sundheim et al .

approximately lO s cfu of bacteria per seed and planted in sand . F. oxysporum 1'. sp . redolens was increased on PD broth for 4 days at 29 '(,'on a rotary shaker . When the plants were I week old, they were carefully removed from the sand and dipped in a conidial suspension of this fungus . The plants were transplanted into potting soil and grown at 18 "C in a growth chamber with a 12 h light 12 h dark cycle . Half strength Hoagland solution was added as needed, and the plants were harvested two weeks later . The plants were then evaluated for wilting . The procedures used to assess the suppression of take-all of wheat (causal agent . G . graminis var . lritici) were as follows . The methods used for fungal oat kernel inoculum preparation were as previously described [26] with the following exceptions . Fungal mycelium grown in PD broth was macerated and used to inoculate sterile oat seed . The final size of the inoculum particles was 0 . 36 to 0 . 71 mm . Bacteria were grown in 100 ml of NBY broth and harvested by centrifugation . Cells washed with 0 . 5 volumes of sterile water were resuspended in l-2 ml of sterile water and then mixed with fungal inoculum . After incubation in a partial vacuum for 12-16 h, the mixture was dried in a sterile airstream . Take-all suppressiveness of bacterial strains was tested by tube assays as described by Weller et al. [25] . We used 20 cm cones and fifty grams of pasteurized soil per cone . Fungal inoculum was mixed with the soil to a final concentration of 1 . 4 mg g -1 . Each experiment contained several treatments with 14 cones per treatment . Plants were maintained in a growth chamber at 16 ° C on a 12 h light 12 h dark cycle . After 4 weeks, plant roots were washed and rated for the severity of root and lower stem infection using the disease index of Weller et al . [25] . In addition, the weight of plant tops were recorded after drying at 100 °C for 24 h . In one experiment, bacteria obtained from root washings were spread on NBY plates with tetracycline to test for the presence of cosmids . RESULTS Identification of chitinase encoding genes

Plasmid DNA was isolated from random clones of the S . marcescens BJL 200 cosmid library and analysed by restriction enzyme digest . More than 90% of the random clones from the library contained inserts in the cosmid vector pLAFR3 . The inserts in the cosmid clones ranged in size from 18 to 28 kb with an average size of 20 . 2 kb . A total of 5686 transductants were isolated and screened for chitinase activity on the overlay plates . Twenty-one clones from the library produced clear zones on chitin-containing medium . These were considered to have cosmids with a chitinase encoding gene . Clearing on the chitin-containing medium was usually visible within 48 h . With some clones the clearing was first obvious 5-7 days after inoculating the chitin overlay plates . All clones containing EcoRI fragments with the chitinase producing phenotype could be divided into two groups . The first group of four had an 18 kb EcoRI fragment ill common, while the second group of seventeen had a 9 . 4 kb fragment in common . Cosmids from both types were purified on CsCI gradients . The cosmid DNA was then digested to completion with EcoRI and re-ligated . The intent of the ligation was to produce cosmids with single EcoRI fragment inserts . The re-ligated cosmids were used to transform HB1OI cells which were screened for tetracycline resistance and white colour on X-gal plates . Transformants were replica-plated to chitin medium . Cosmids from chitinase positive clones were isolated by the miniprep protocol [10] .

Molecular cloning of two chitinase genes 1

2

487 3

5

7

23 •I

9.4 6.6 4.4

2.3 2. 0

FIG . 1 . Southern hybridization analysis of EcoRI digested DNAs probed with radiolabelled ALES 1 . Lane 1 lambda DNA digested with Hind III . Lane 2 pBR325 digested with EcoRI . Lane 3 pLES1 digested with EcoRl . Lane 4 pLES2 digested with EcoRI . Lane 5 pBR325 digested with EcoRl and probed with pLES4 . Lane 6 pLES I 1 digested with EcoRI and probed with pLES 1 . Lane 7 pLES2 digested with EcoRl and probed with pLESI .

Analysis with restriction enzymes indicated that cosmid pLES1 had a single 9 . 4 kb EcoRI insert . Cosmid pLES2 had a single 18 kb EcoRl insert . Both had a chitinase positive phenotype . These two cosmids were mobilized from E. coli strain HB101 into the strain Ill and Ps fluorescens strain NRRL B-15135 by the triparental mating procedure of Ditta et al . [3], with pRK2013 as helper plasmid . To facilitate further analysis, the cosmid inserts of pLESI and pLES2 were subcloned into plasmid pBR325 [1] to produce respectively plasmids pLES3 (containing the 9 . 4 kb fragment) and pLES4 (containing the 18 kb fragment) . Characterization of the two chitinase encoding plasmids

Plasmid pLES3 was nick translated and used as a probe in Southern hybridization experiments to determine the relationship between the two EcoRI fragments of 9 . 4

488

L . Sundheim et al.

(a) RV D S RV RIIB S B V S S RV IB IRI I I I I I 1 1 I

2 kb I 1

(b)

RI

Bg

RV

S

D

RV S RV

S RV

S S

RV B

8

BBg

D

1 1 1 1 I 1, I I I 1 I I I I I

IRI

1 1

FIC . 2 . Restriction maps of the 9 . 4 kb insert (a) from plasmid pLESI and the 18 kb insert (b) from plasmid pLES2 . Abbreviations : B, BamHI ; Bg, BglI I ; D, Dral ; RI, EcoRI ; RV, EcoRV : S, Sall .

TABLE

2

The effect of culture filtrates of Pseudomonas fluorescens strain NRRL B-15135 with and without the chitinase encoding cosmid pLESI on the growth of

Fusarium oxysporumf. sp . redolens germ

tubes

Germ tube length, pm Culture filtrate None Medium only NRRL B-15135 NRRLB-15135+pLESI

Average'

SD`

213 451 466 94

96 174 127 62

'Average of 50 germ tubes per treatment . 'Standard deviation .

and 18 kb respectively . Figure 1 shows results from one representative hybridization following EcoRI-digestions of plasmids pLES3 and pLES4 . The 18 kb EcoRI fragment showed no homology with the 9-4 kb fragment . Whole cell DNA from S. marcescens digested with EcoRI or EcoRV was also probed with radiolabelled pLES3 . The 9-4 kb EcoRI fragment hybridized to the expected fragments in restriction digests of whole cell DNA from S . marcescens (data not shown) . When restriction enzyme maps of the two chitinase encoding fragments of S . marcescens were compared, no similarity between the 9 . 4 kb insert in pLES3 and the 18 kb insert in pLES4 was apparent (Fig . 2) . Effect of the chitinase producing bacteria on plant pathogens in vitro and in vivo . Strains Ill and Ps fluorescens strain NRRL B-15135, both containing cosmid pLESI were grown separately for 48 h on NBY medium with nalidixic acid, rifampicin, and tetracycline . The bacteria were removed by centrifugation and the supernatant was filtersterilized . The culture filtrate was mixed with water agar at 50 ° C and allowed to solidify . Culture filtrate from the chitinase-producing bacterial strain NRRL B-15135 containing cosmid pLES 1 appeared to be fungistatic to two Fusarium form species tested . Significant reduction in germ tube growth was noted for both fungi (Tables 2, 3) . Hyphal growth of the two plant pathogens Rhizoctonia solani and Magnaporthe grisea was also determined in the presence of P.Huorescens strain B-15135 and strain Ill streaked

Molecular cloning of two chitinase genes

489 TABLE 3

The effect of culture filtrates of Pseudomonas fluorescens strain NRRL B-15135 with and without the chitinase encoding cosmid pLESI on the growth of Fusarium oxysporum f . sp . conglutinans germ tubes

Germ tube length, µm Culture filtrate

Average'

SD`

188 334 239 97

77 175 111 67

None Medium only B-15135 B-15135+pLESI 'Average of 50 germ tubes per treatment . "Standard deviation .

TABLE 4 Control of Fusarium oxysporumf. sp . redolens on radish by a rhizobacterium (strain Ill) and Pseudomonas fluorescens strain .NRRL B-15135, with and without the chitinase encoding cosmid pLESI

Bacterial strain

Cosmid

Fungus

Average disease index-

B-15135 B-15135 III I11 LSD P=0 . 05

pLESI pLESI

+ + + + +

0 8.0 8.9 6 .0 7.7 7.5 1.3

'Values are average of four plants per treatment in two different experiments . The disease index was calculated from the number of dead plants at the end of both experiments : 0= no dead plants and 10 = all plants dead .

across the plate . Cosmid-containing strains showed greater inhibition of the two fungi than the parental strains (data not shown) . The presence of cosmid pLES 1 improved the effect of the Ps fluorescens strain NRRL B-15135 in the control of F. oxysporum sp . redolens, but with strain 111, no effect of the cosmid was apparent (Table 4) . Cosmid pLES1 had no significant effect on the take-all suppressive properties of strains NRRL B-15135 and Ill (data not shown) . When 100 random fluorescent colonies were analysed at the end of the experiment, all were sensitive to tetracycline (Table 5) . This indicates that the cosmid was lost from the take-all suppressive strains in the course of these experiments . DISCUSSION We have identified two regions with chitinase encoding activity in the S. marcescens genome . The two regions are different as determined by restriction enzyme analysis and

490

L . Sundheim et al. l'ABLE

5

Growth on antibiotic-containing media of single colonies isolated from wheat plant roots

Treatment' Sterile water NRRL B-15135 NRRL B-15135+pLESI (Tc')

No . of fluorescent cfu g of root tissue

Growth on medium containing Nal" Nit] +Tc"

0( < 1 x 10') 4 . 5 x 10°

100

0

9 . 6 x 10°

100

0

'Bacteria were recovered from plant roots growing in soil containing G. graminis oar tritici inoculum which had been treated with sterile water or bacterial suspensions . ° 100 fluorescent isolates recovered from each treatment were tested for growth on NBY medium containing Nal (nalidixic acid) or Nal and Tc (tetracycline) .

Southern hybridization . One region may be analogous to the 9 . 5 kb EcoRI fragment previously identified [5], while the 18 kb fragment appears to be a different chitinase encoding region of the S. marcescens chromosome . Although the presence of plasmid pLESI in strain NRRL B-15135 inhibited the growth of plant pathogens in vitro and suppressed disease caused by F . oxysporum f. sp . redolens, it was less effective in controlling G . graminis var . tritici, the causal agent of take-all of wheat . Cosmid pLES I was not maintained in strain NRRL B-15135 during the course of the take-all experiment . This may explain the greater effect of the chitinase encoding cosmid in the 3-week-long Fusarium experiment . It is possible that improved control of take-all might be achieved if the plasmid could be stably maintained . Fungistatic effects ofE. coli strains with chitinase encoding cosmids have been noted previously [6] . The rapid loss of the pLAFR3-derived cosmid pLES 1, in Ps fluorescens and in the Gram negative strain Ill, makes it impractical for disease control . Other cosmids or plasmids should be tried to identify more stable extrachromosomal elements as carriers for the chitinase encoding EcoRI fragment . Homologous integration into the chromosomal DNA of the biocontrol strain is another approach that should be explored . REFERENCES 1 . BOLIVAR, R ., RODRIGUEZ, R. L ., GREENE, P. J ., BETLACH, M . C ., HEYNEKER, H . L ., BOYER, H . W., GROSA, J., & FALEOW, S . (1977) . Construction and characterization of new cloning vehicles . II . A multipurpose cloning system . Gene 2, 95-113 . 2 . BOLLER, T ., GEHRI, A ., MAUCH, F . & VOGELI, U . (1983) . Chitinase in bean leaves : Induction by ethylene, purification, properties, and possible function . Planta 157, 22-31 . 3 . DITTA, G., STANFIELD, S ., CORBIN, D . & HELINSKI, D . R . (1980) . Broad host range DNA cloning system for Gram-negative bacteria : Construciton of a gene bank of Rhizobium melioloti . Proceedings of the National Academy of Science, USA 77, 7347-7351 . 4 . ELANGO, N ., CORREA, J . U . & CABIB, E . (1982) . Secretory character of yeast chitinase . Journal of Biological Chemistry 257,1398-1400 . 5 . FUCHS, R . L ., MCPHERSON, S. A. & DRAHOS, D . J . (1986) . Cloning of a Serratia marcescens gene encoding chitinase . Applied and Environmental Microbiology 51, 504-509 . 6 . JONES, J . D . G ., GRADY, K . L., SUSLOw, T . V . & BEDBROOK, J . R . (1986) . Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens . The EMBO Journal 5, 467-473 . 7 . KEEN, N . T ., DAHLBECK, D., STASKAWICZ, B . & BELSER, W. (1984) . Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli . Journal ofBacteriology 159,825-83 l . 8 . KING, E . O ., WARD, M . K. & RANEY, D. E . (1954) . Two simple media for the demonstration of pyocyanin and fluorescin . Journal of Laboratory and Clinical Medicine 44, 301-307 .

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9 . KLOEPPER, J . W . & SCHROTH, M . N . (1978) . Plant growth promoting rhizobacteria on radishes . In Proceedings, IV International Conference on Plant Pathogenic Bacteria . Angers, France, Vol. 2, pp . 879-882 . 10 . MANIATIS, T ., FRITSCH, E . F . & SAMBROOK, J . (1982) . Molecular Cloning: A Laboratory Manual . Cold Spring

Harbor Laboratory, Cold Spring Harbor, New York . 11 . MANKAU, R . & DAS, S . (1969) . The influence of chitin amendment on Meloidogyne incognita . journal of Nematology 9, 192-19 7 . 12 . MITCHELL, R . & ALEXANDER, M . (1962) . Microbiological processes associated with the use of chitin for biological control . Soil Science Society of America, Proceedings 26, 556-558 . 13 . MONTREAL, J . & REESE, E . T . (1969) . The chitinase of Serratia marcescens. Canadian Journal of Microbiology 15,689-696 . 14. MUZZARELLI, R . A. (1977) . Chitin . Pergamon Press, New York . 15 . PAPAVIZAS, G . C . & DAVEY, C . B . (1960) . Rhizoctonia disease of bean as affected by decomposing green plant materials and associated microfloras . Phytopathology 50, 516-522 . 16 . PEGG, G . F . & VESSEY, J . C . (1973) . Chitinase activity in Lycopersicon esculentum and its relationship to the in vivo lysis of Verticillium albo-atrum mycelium . Physiological Plant Pathology 3, 207-222 . 17 . POPLAWSKY, A . R ., PENG, Y . F . & ELLINGBOE, A . H . 1988 . Genetics of antibiosis in bacterial strains suppressive to take-all . Phytopathology 78 : 426-432 . 18 . REID, J . D . & OCRYDZIAK, D. M . (1981) . Chitinase-overproducing mutant of Serratia marcescens. Applied and Environmental Microbiology 41, 664-669. 19 . SNEH, B . (1981) . Use of rhizosphere chitinolytic bacteria for biological control of Fusarium oxysporum f. sp . dianthi in carnation . Phytopathologische Zeitschrift 100, 251-256 . 20 . SNEH, B ., ACAMI, O . & BAKER, R . (1985) . Biological control of Fusarium-wilt in carnation with Serratia liquefaciens and Hafnia alvei isolated from rhizosphere of carnation . Phytopathologische Zeitschrift 113, 271-276 . 21 . SNEH, B ., KATAN, J . & HENIS, Y . (1971) . Mode of inhibition of Rhizoctonia solani in chitin-amended soil . Phytopathology61, 1113-1117 . 22 . STASKAWICZ, B., DAHLBECK, D ., KEEN, N . & NAPOLI, C . (1987) . Molecular characterization of cloned avirulence genes from race 0 and race I of Pseudomonas syringae pv . glycinea . Journal of Bacteriology 169, 5789-5794 . 23 . VESSEY, J . C . &PEGG, G . F . (1973) . Autolysis and chitinase production in cultures of Verticillium albo-atrum . Transactions of the British Mycological Society 60, 133-143 . 24 . VIDAVER, A . K . (1967) . Synthetic and complex media for the rapid detection of fluorescence of phytopathogenic pseudomonads : Effect of the carbon source . Applied Microbiology 15, 1523-1524 . 25 . WELLER, D . M ., ZHANG, B.-X . &COOK, R . J . (1985) . Application of a rapid screening test for selection of bacteria suppressive to take-all of wheat . Plant Disease 69, 710-713 . 26 . WILKINSON, H . T ., COOK, R . J . & ALLDREDGE, J . R . (1985) . Relation of inoculum size and concentration to infection of wheat roots by Gaeumannomyces graminis var . tritici . Phytopathology 75, 98-103 .