Competitiveness and persistence of strains of rhizobia for faba bean in acid and alkaline soils

Pergamon

0038-0717(94)00145-6

Soil Bid. Biochem. Vol. 27, No. 415, pp. 617623, 1995 Copyright 0 1995 Elkier Science Ltd Printed in Great Britain. All rights reserved

0038-0717/95$9.50+0.00

COMPETITIVENESS AND PERSISTENCE OF STRAINS OF RHIZOBIA FOR FABA BEAN IN ACID AND ALKALINE SOILS JANINE M. CARTER,’ JAMES S. TIEMAN* and ALAN H. GIBSON’* IDepartment of Agriculture, Victorian Institute for Dryland Agriculture, Private Bag 260, Horsham, Vie. 3401, Australia and *CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia ecological aspects of the rhizobial inoculation of faba beans were examined, namely their persistence in acid soils and their competitiveness in soils with high naturally-occurring populations of rhizobia. Of 8 Rhizobium leguminosurum bv. uiciae strains introduced into 3 acid soils (pH range 4.8-5.7) only 3 consistently maintained soil populations in excess of 100 g-i 2 years after planting. This poor persistence was reflected in nodulation scores and plant growth of uninoculated faba bean sown 2 years after the initial introduction of inocula. Only plants in plots originally inoculated with WSM1014 had yields that were comparable to that of plots sown with freshly-inoculated (commercial strain SU303) seed. In the same area in southwest Victoria, soils of 11 commercial faba bean crops that had been inoculated with SU303, were sampled 1 year later and the total number of rhizobia in the soils were compared with those in soil from adjacent paddocks with no grain legume cropping history. Below pH 6, numbers in the sown soils were low, with little or no increase over numbers in unsown soil. Above pH 6, the numbers in the unsown soil varied from “not detectable” to l(r g-i, and the sown soils showed a variable increase. In only 3 inoculated and 2 uninoculated soils (pH 6.08-8.25) did rhizobia equal or exceed 10) g-‘. Serological examination of isolates from MPN plants indicated poor establishment of the inoculant strain. In conclusion, the persistence of R. leguminosarum bv. viciae strains in acid soils was poor, however strains with improved persistence may be selected. Five potential inoculant strains were introduced as inoculants on faba bean seed into 7 neutral-to-alkaline soils in the Northern Wimmera, which possessed high populations of competitive native strains. At 12 wk, all plants, including the uninoculated controls, were well-nodulated. At 16 wk, serological examinations indicated only a low level of infection by the inoculant strains. At harvest, there were no yield increases due to inoculation. At one site, isolates from nodules in uninoculated plots were serological identical to one of the inoculant strains, but the application of molecular biology techniques showed they were not the inoculant strain. Another strain showed a loss of infectivity in the peat inoculant. These results demonstrated the difficulty of demonstrating a response to inoculation in the presence of a substantial native population.

Summary-Two

INTRODUCTION

The introduction of an improved commercial faba bean (Viciufuba) inoculant strain (SU303) in 1990 has enabled the expansion of the crop into the acid soils of southwest Victoria (Carter et al., 1994), South Australia (Silsbury, 1991) and Western Australia (K. Siddique, pers. commun.). However, once introduced to these soils, it is not known whether this strain survives or persists in sufficient numbers to nodulate subsequent faba bean crops adequately. In Victoria, the majority of faba beans are currently grown in the alkaline soils of the Northern Wimmera. Seed of faba bean, field peas (Pisum sutivum), lentils (Lens culinaris) and vetch (Viciu spp) are rarely inoculated in these soils but they are often well-nodulated. The symbiotic effectiveness of the naturalized strains, compared to the faba bean commercial inoculant, SU303, is unknown. In France, high numbers of effective Rhizobium Ieguminosarum *Author for correspondence. SBB 27rCI--P

bv. oiciue occur in alkaline soils, as compared to soils with pH < 5.5, where numbers were mostly undetectable (Amarger, 1988). Therefore, it was important to examine pH effects under Australian conditions. The persistence of 8 strains of R. leguminosarum bv. uiciue, was examined 2 years after introduction to 3 acid soil sites. We also report a survey of the persistence of the commercial faba bean strain after 1 year in 11 acid soils of southwest Victoria. Results of field inoculation trials at 7 sites, involving 5 strains of R. Ieguminosarum bv. viciae, are reported for the alkaline soils in the Northern Wimmera of Victoria.

MATERIALS

AND METHODS

The southwest region of Victoria generally receives more than 550 mm rainfall per annum. The soils are typically duplex loams over clay, range from pH(H20) 4.5 to 6.5 (O-10 cm), and periodic waterlogging in winter and spring is common. The Northern Wimmera region in northwest Victoria generally receives 617

618

Janine

M. Carter

between 350-500 mm rainfall per annum. The majority of soils are alkaline and range from pH(H20) 6.5 to 8.5 (&IO cm). Grey and brown self-mulching clays are the dominant soil type (40%) followed by red duplex soils (20%) (Badaway, 1982). Persistence of rhizobia in acid soils

Experimental sites were at Balmoral, Lake Bolac and Hamilton (Fig. I), where 8 R. leguminosarum bv. viciae strains were introduced on faba bean seed in 1989. The trial layout was a randomized block design, with 4 replicates, each treatment plot consisting of 8 rows (17 cm apart), 30 m long (Carter et al., 1994). These plots were sown to wheat in 1990. Immediately prior to sowing faba beans in 1991, 10 soil cores (2.5 cm dia) from the top 10 cm were collected from each replicate for all treatments at Balmoral and for 3 treatments (uninoculated, SU303, CC328) at Lake Bolac and Hamilton. For each site, the 10 cores of each replicate were bulked, air-dried and stored at 4°C for 4 wks. The resident rhizobial population was enumerated by the most probable number (MPN) technique, using V. hirsuta as the test host (Brockwell, 1980; Marshall et al., 1993). “Sprayseed” (250 g paraquat a.i. ha-’ + 150 g diquat a.i. ha-‘) was applied at Hamilton after sampling and prior to sowing; “Simatox” (2 kg simazine a.i. ha-‘) was sprayed post-seeding at all sites. “Fusilade” (106 g fluazifop-p a.i. ha-‘) was applied at Balmoral and Lake Bolac on 18-l 9 June to control grass weeds. All plots were sprayed in June (Balmoral, Lake Bolac) or July (Hamilton) and September with “Dithane M-45” (2 kg mancozeb a.i. ha-‘) to control ascochyta blight (Ascochyta fabae Speg.-anamorph of Didymella fabae Jellis and Punith) and chocolate spot (Botrytisfabae Sard. and B. cinerea Fr.). Untreated seed of V.fuba cv. Fiord was then sown at 120 kg seed ha-’ with 17 kg P ha-’ as double superphosphate, over the plots established in 1989. Seed inoculated with the commercial inoculant (strain SU303, obtained from Inoculant Services Australia Pty Ltd, “Teangi”, Bethanga, Victoria) was also sown. The seed was slurry-inoculated with water immediately prior to sowing at the commercially recommended rate. The seeder box and seed tubes were cleaned with 70% ethanol at each change of site. Sowings were completed between 2-7 May. Plant emergence was determined in July by counting plants in two 1 m* quadrats per plot. Nodulation of 5 plants plot-’ was assessed at 8-l 1 wk after sowing, and during flowering (18-19 wks) using arbitrary ratings for both number and size of nodules (Carter et al., 1994). The two values were multiplied to provide a single score per plant, representing an index for total nodule tissue present. Each plot was assessed visually for crop height, vigour and colour on an arbitrary l-1 0 scale at 25-26 wk after sowing (pod-filling) and the grain harvested at 35-37 wk (3-16 January).

el al.

Persistence

of rhizobia

in commercial faba bean crops

Eleven paddocks from 11 properties located in southwest Victoria were surveyed in June 1991 (Fig. 1). Two replicates of 10 soil cores (2.5 cm dia) from the top 10 cm were collected where inoculated (SU303) faba beans had been grown 12 months previously. For each property a similar, unreplicated sample, was collected from an adjacent paddock which had never been sown to grain legumes. Each replicate was bulked, and all samples were air-dried and stored at 4°C for 4 wk prior to pH analysis (HZ0 and CaC12). The rhizobial population sizes were determined by the MPN method described above. Competitiveness of introduced strains

The sites, with neutral-to-alkaline soil pH, at Lillimur South, Miram West, Dooen, Warracknabeal and Charlton had no grain legume cropping history. Two similar sites, located at the Wimmera Research Station (WRS, Dooen) and Charlton, had grown field peas for which inoculant had been used at least once. The soil types were all grey or brown self-mulching clays apart from the sites at Charlton (no legume history) and Miram West, which were red duplex soils; the latter was particularly sodic. Immediately prior to sowing in 1990, 10 soil cores (2.5 cm dia) from the top 10 cm were collected at each site (Fig. 1) and treated and tested as described above. All sites received a knockdown herbicide after soil sampling and prior to sowing and “Simatox” (600 g simazine a.i. ha-‘) tank-mixed with “Gesaprim” (400 g atrazine a.i. ha-‘) post-seeding. “Fusilade” (106 g fluazifop-p a.i. ha-‘) was sprayed at all sites on 18-31 July to control grass weeds. All plots were sprayed in August and October with “Dithane-M45” (2 kg mancozeb a.i. ha-‘) to control ascochyta blight (A. fabae) and chocolate spot (B. fabae and B. cinerea). Peat inoculants of five strains of R. leguminosarum bv. viciue, including the commercial inoculant strain SU303 containing lo9 colonies g-’ peat were mixed with 0.5% methyl cellulose and used to slurryinoculate seed at recommended rates immediately prior to sowing. Uninoculated controls were included at all sites. A randomized block design with 4 replicates was used with 8 rows (17 cm apart) 30 m long per plot (15 m at WRS, Dooen). The seeder box and seed tubes were cleaned with 70% ethanol at each change of inoculant. Sowing was between 30 April and 29 May using V.faba cv. Fiord at 120 kg seed ha-’ with 10 kg P ha-’ as double superphosphate. Plant emergence was measured in early June and nodulation was assessed during flowering (12 wk after sowing) as described previously. At 16 wk, 5 plants plot-’ were removed for the recovery of inoculant strains. Nodules were surface sterilized (Gault et al., 1973). the juice from crushed nodules plated onto YMA and selected single colony isolations stored at 4°C. Each plot was harvested for grain yield at 28-30 wk (3-19 December).

619

Ecology of inoculant faba bean rhizobia

3 &SHAM RB H 0 Balmoral L D v s 0 Hamilton 0 LaJceBolac ELBOURNE 0

A

Fig. 1. Location of trial (0) and farm survey sites (V, Lake Bolac; S, Streatham; D, Tatyoon; L, Vasey; N, Wamcoort; C, Wombelano; A, Duvemey; H, Nurrabiel; R, Noradjuha; B, McKenzie Creek; 0, Berrybank) in southwest Victoria and of the inoculant competition trial sites (1, Lillimur South; 2, Miram West; 3, Dooen; 4, Warracknabeal; 5, Charlton; 6, WRS, Dooen; 7, Charlton) in the Northern Wimmera.

Serological identiJcation

of isolates

Freshly-grown cultures were suspended in 0.85% saline, centrifuged (6000 rev min-‘, 15 min) and the pellet added to wells in immunodiffusion plates, adjacent to wells containing standard antigen of the strain under examination (Dudman, 1977). The plates were held at 4°C for 3-4 days before assessment. Statistical

treatment

Nodulation score and yield data were subjected to analyses of variance. RESULTS

Persistence of rhizobia in acid soils

The

populations

of

introduced

strains

of

R. leguminosarum bv. viciae in soils at 3 sites sown

2 years previously with inoculated faba bean, indicated

very poor recovery of the commercial inoculant strain, SU303, and one other strain considered to have good symbiotic capability with faba beans, CC328 (Table 1). At the one site where all 8 treatments were sampled, only 3 strains (NZP5225, WSM1014 and CC322) were greater than 100 rhizobia g-r soil. There was a high degree of variability, with R. leguminosarum bv. viciae present in 3 of the 4 replicates in the NZP5225 and WSM1014 treatments, and 1 replicate of the CC322 treatment. Plant establishment in the plots was 30 plants m-2 (25-36 plants m-*), an acceptable commercial population. Nodulation scores at 8-11 wk, and again 18-19 wk after sowing (Table 2), reflected the results of the MPN assays (Table 1). The plants in the WSM1014 and NZP5225 plots, along with the plants sown with freshly-inoculated (SU303) seed, consistently achieved the highest scores across the 3 sites.

620

Janine M. Carter et al.

Table I, Recovery of unidentified R. legumrnosarum bv. twrae (MPN) from 3 sites. sown with inoculated faba beans 2 years previously (bacteria g-’ soil)

Inoculant strain Unmoculated su303 CC328 su391 NZP5474 NZP5471 CC322 NZP5225 WSM1014

Balmoral,

soil

Lake Bolac. soil

pH(HlO) 5.7 <2.2 3.2

pH(H>O)5.0 2.0 <2.2

I.1 0.7 <2.2 0.5 II0 227 192

Hamilton,

ND ND ND ND ND ND

*ND = not determined

Site*

_

Stram

soil

pH(H>O)4.8 < 2.2 <2.2
I.4 ND* ND ND ND ND ND

Table 3. Grain yield (t ha-‘) of faba beans in plots sown to inoculated faba beans 2 years previously, or freshly-inoculated (SU303) in acid soils

Balmoral

-_ Lake Bolac

0.82d 0.78d 0.67d 0.91bcd 0.9lbcd 0.89cd 1.55a 1.12bc 1.41a

0.37d I.llab 0.49cd 0.61cd 0.45cd 0.79bc I .22a I .08ab I .33a

0.47b 0.36bc 0.34bc 0.48bc 0.70b 0.53bc I .29a 0.68b I .46a

LSD(P = 0.05)

0.30

0.41

0.41

*Within a site, yield values followed significantly different (P= 0.05).

Occasionally, nodulation of faba bean in previously inoculated plots was greater than that of the uninoculated plots, despite an inability to detect rhizobia by MPN assays prior to sowing. A visual rating of the plots for biomass at 25-26 wk after sowing reflected the nodulation scores (data not shown). At harvest, only the WSM1014 treatment consistently achieved yields comparable to those sown with freshly-inoculated seed (Table 3). The other strain present in moderate numbers at the start of the season, NZP5225, and which showed good early nodulation, was inferior to WSM1014 and the freshly-inoculated treatment at two of the sites. Any improvement in yield of plants grown in previously inoculated plots, apart from WSM1014 and NZP5225, was marginal, and very variable across the three sites. of rhizobia in commercial,fhba

bean crops

Eleven paddocks, ranging in pH(H20) from 4.83 to 8.25 (CaC&: 4.1g7.78) showed a wide range in the R. leguminosarum bv. viciae population 12 months after farmer sowing ofinoculated faba beans (Table 4). At 2 of the 7 sites with pH(H20) < 6, the population was the same size as that taken from adjacent unsown paddocks, while at another site, a 300-fold increase was observed (Table 4). Above pH 6, 3 sites showed a moderate-to-high population of rhizobia in the unsown soil, and in only one of these sites (site 0), the Table 2. Mean nodulation

of introduced

strains

All soils were of neutral to alkaline pH and contained substantial populations of R. leguminosarum bv. viciae. Consequently, only a very low proportion of the nodule population was formed by the inoculant strains (Table 5). One strain, CC328, appeared to be competitive with local rhizobia at 2 sites (> 25% of nodules), but serological analysis of nodules from the uninoculated controls from the Charlton sites showed significant cross-reaction with CC328 antisera. Examination of these isolates using polymerase chain reaction (PCR) technology with the

scores* for faba beans growing in plots sown to inoculated sown with freshly-inoculated (SU303) seed

faba beans 2 years previously,

Weeks 8-1 I Strain

by the same letter were not

site with the highest pH, was the increase in population size substantial. A large increase, to 10’ g-‘, was observed at site H, pH 6.1. Isolations were made from nodules of plants in the MPN series from 2 sites, and tested for serological affinity with antisera to the commercial inoculant, strain SU303. From site R, where the populations in both sown and unsown soil were of the order of lo4 g-‘. no isolate gave a reaction of identity. However, 13 isolates (25%) from both sown and unsown soil of other sites cross-reacted with the antiserum. From site N, with many fewer nodules available for isolation, 3 of the 7 isolates from the sown area gave a reaction of identity with SU303 antiserum, whereas none of the 5 from the unsown soil reacted. Competitiveness

Persistence

Hamilton

Uninoculated CC328 NZP5471 NZP5474 CC322 su303 (1989) su303 (1991) NZP5225 WSMlOl4

or in plots

Weeks 18-19

Balmoral

Lake Bolac

Hamilton

Uninoc. CC328 NZP5471 NZP5474 CC322 su391 SU303 (1989) su303 (1991) NZP5225 WSMIOI4

0.8 1.0 1.3 1.4 I.5 1.9 I.1 2.1 2.3 2.6

0.2 0.2 0.3 0.7 0.4 0.5 0.4 2.2 1.4 I.2

0.1 0. I 0.2 0 0.1 0.5 0.1 1.5 0.8 I.1

3.1 I.8 2.0 1.9 2.9 3.3 2.2 3.4 3.2 3.4

1.0 0.9 0.5 0.7 I .o 1.2 0.9 2.4 1.3 2.7

LSD(P=O.OS)

1.0

0.9

0.8

NSt

I.2

Balmoral

Lake Bolac

*Scoring was on a t&4 basis for nodule number and &3 for nodule size, the final index being the multiplicand tNS=not significantly different (P=O.O5).

Hamilton 0.3 0.7 0.3 0.1 0.4 I .o 0.4 22 1.7 0.9 1.0 of the two scores.

Ecology

of inoculant

faba bean rhizobia

621

ABCDEFGH

Fig. 2. PCR amplification of R. leguminosarum bv. uiciae DNA from strain CC328 (lane B), from isolates from uninoculated plots (lanes C-E) and from isolates from plots inoculated with strain CC328 (lanes F-H). The amplification products were generated from freeze-thaw lysate templates, using RPOl as primer. The standards (lane A) are bacteriophage SPP-1 DNA digested with EcoRl.

nif-directed primer, RPOl (Richardson et al., 1995) revealed that the presumptive CC328 isolates from the uninoculated plots were not strain CC328 (Fig. 2); this was confirmed by restriction fragment length polymorphism analysis using pRt587 as a probe (Demezas et al., 1991) (data not shown). This observation casts doubt over the apparent superior competitiveness of strain CC328 at these sites.

The poor competitiveness of WSM937 may have been due to the existence of non-invasive mutants in the applied inoculant. Although the inoculants contained in excess of lo9 rhizobia g-’ immediately prior to application, and packets containing WSM937 had maintained their viability after 6 months storage at 4°C 23 out of 24 single colony isolates failed to nodulate V. hirsuta growing under bacteriologically-

622

Janine M. Carter et cl/. Table 4. Populations of R. kgunritwsurum bv. wux (number g ’soil) m the sods of I I commercial faba bean crops inoculated Yvith SU303 I2 months after sowing (mean of 2 replicate samples). and in adiacent unsown soil Number

DH Site*

H>O

CaClz

V S D L N C A

4.83 S.18 5.28 5.35 5.62 5.85 S.85

4.10 4.33 4.50 4.70 4.38 4.78 4.4s

H R B 0

6.08 6.23 6.83 8.25

5.20 4.85 5.75 7.78

*See Fig. I. tvl duplicates :v2 duplicates

I.101 10.10’ 259 50.10’

site Lillimur S.t Miram W.t Dooent WRS. Dooen: Warracknabealt Charltont Charlton:

4.7 I 7 17 7.9 3.4 I.4 X.8

Increase

2 8 <2 t2 48 <2 9

300 x IOX 0 20X 0 IOX IOX

8 11.10’ 160 1.10’

125x 0 0 50X

consistent persistence, albeit at only lo&200 rhizobia g-’ soil. Nodulation of plants sown into plots previously inoculated with these 2 strains reflected their persistence and was comparable to that of plants grown from freshly-inoculated seed. Hence, strains WSM1014 and NZP5225 (syn. NA526) must be regarded as being more tolerant ofacidic soils than the other strains examined. Although strain NZP5225 showed similar persistence to WSM1014, and a comparable level of nodulation early in plant growth, yield at final harvest tended to be inferior to that achieved in the WSM 10 14 treatment. This is consistent with the lower effectiveness observed in glasshouse studies with this strain (A. H. Gibson, unpubl. data). The poor persistence of the commercial inoculant strain in acid soils was also indicated by the survey of 11 soils sown with seed inoculated by farmers. In only 1 of 7 soils below pH(H20) 6 were the numbers of R. leguminosarum bv. viciae in excess of 100 g-‘. even though in most cases there was a considerably higher population relative to that from an adjacent unsown area. Whether these numbers are adequate to achieve good nodulation is doubtful, although in the strain trial (Tables l-3) 200 rhizobia g-’ at the start of the season provided reasonable nodulation.

There has been a marked increase in the cultivation of faba beans in the acid soils of southwest Victoria, especially since the introduction of an improved inoculant (SU303) for the species (Silsbury, 1991; Carter Pt d., 1994). In 1989, inoculation with this strain, markedly improved yields at 6 sites, however 2 years later. this strain showed very poor persistence at 3 sites examined, as did CC328 and 4 other strains previously used as inoculants. Only 2 strains showed

lmtial population (IO?)

’sod

varied, 7 and 242; pH values the same. varied, 38 and ~2: pH 5.65 and 6.05.

DISCUSSION

straw

e

UllSOWIl

628 84(v I )t <2 44 34 17(v2): 97

controlled conditions in test tubes. Similar examination of the mother culture used to prepare the inoculant showed only 2 out of 25 single colony isolates failing to nodulate this host. Concurrent examination of a peat inoculant containing strain SU303 showed a good correspondence between total viable count on plates and counts based on MPN procedures using V. hirsuta as test host. Across the 7 sites, the plants established at 21 plants m ~? (17-25 plants m- ‘). At flowering, the nodulation of all treatments at the 7 sites was very good, with no significant improvement due to inoculation (date not shown). At the final harvest, there was no effect of inoculation on grain yield.

Table 5. Recovery of moculant

of rhlzobla

Inoculated

of R. /~~grrr?lino.\urunl bv. r~iuar from 7 field sites sown I6 wk previously by gel immunodiffusion on 35-40 isolates

wth faba beans. as determined

% Isolates identified pH(H0 7x 77 8.1 x.3 x.3 6.6 7.x

su.303 19 II 0 0 IX 2s 0

C(‘i7X II 30 3 II 32 74 63

WSM937 0 5 7 0 0 0 0

su391

NZZP5472

Uninoc. controlsij

0 7 0 I6 3 Y 21

0 I7 3 0 2 23 3

ND1 6% ND ND ND 26% 30%

‘Initial soil population of rhlzobia determined by MPN counts on soil taken at the time of sowing. tNo prewous grain legume crop. $Inoculated iield pea crop grown previously. $Proportion givmg reaction of Identity wth CC328 antiserum. no posltlvc reactions wth antisera to other strams. ’ ND = not determined.

623

Ecology of inoculant faba bean rhizobia

The poor recovery of the inoculant rhizobia from the nodules on plants grown in soils with higher pH (Table 5) probably reflected the high populations of naturally-occurring rhizobia able to nodulate faba beans. Five of these sites had no history of cultivation of field peas, faba beans or any other crop likely to be nodulated by R. feguminosarum bv. viciae. This level of competition provides great difficulty when attempting to analyse any potential benefits from inoculation. Analysis of the symbiotic capability of naturally-occurring population with faba beans is in progress to assess whether improved inoculant technology is required to improve yields. There are numerous examples of inoculant strains losing invasiveness, or symbiotic effectiveness (R. J. Roughley, pers. commun.). A recent example involved R. leguminosarum bv. viciae strain TAlOl which was found to be non-invasive in commercial inoculants. That example, and the results presented here for strain WSM937, are interesting in that both suggest that non-invasive mutants can multiply from a low level in the stock culture to eventually dominate a peat culture. Why a mutation relating to invasiveness can provide mutants with a strong competitive edge in culture is an interesting question and justifies closer examination. Also of importance to the inoculant industry is the development of a quality control procedure system, whereby low levels of mutations in a culture can be detected before release to retail outlets. A further question is whether some strains are more likely to mutate for invasiveness, and symbiotic effectiveness, than other strains? In conclusion, the tolerance of R. leguminosarum bv. viciae strains to low pH should be a selection criterion for faba bean inoculants if the crop is to be grown in the low pH soils of southern Australia. The variation in tolerance indicated that selection for this character was feasible, and provides scope to understand the underlying basis of this tolerance. For alkaline soils, the occurrence of large naturally-occurring populations of R. leguminosarum bv. viciae, even where “pea-type” legumes had not been grown previously, reduced nodulation by inoculant strains applied by conventional means. A significant problem to be resolved before undertaking research to overcome this competitive situation is the determination of the symbiotic capability of the naturally-occurring population. The reported loss of invasiveness in an inoculant strain is disconcerting when such mutants build-up from a low level to dominate a peat culture stored at 4°C. The question remains as to whether symbiotic genes in R. leguminosarum biovars have a naturally high mutation rate, and if so, to what extent are there differences between strains. The challenge is to devise screening procedures to either restrict the inclusion of, or to detect, such strains in inoculants.

Acknowledgements-We thank the farmers in southwest Victoria who allowed us to sample soil on their properties (N. Valiance, Lake Bolac; A. Sim, Streatham; B. Doery, Tatyoon; R. Lyons, Vasey; T. and S. Noble, Warncoort; B. Close, Wombelano; R. Alexander, Duvemey; R. Hale, Nurrabiel; G. Rethus, Noradjuha; W. Bothe, McKenzie Creek; D. O’Dwyer, Berrybank) and our site co-operators (C. Hindhaugh, Balmoral; J. Malin, Lake Bolac; R. Crouch, Lillimur South; G. Meyer, Miram West; J. Molyneaux, Dooen: C. Muller. Warracknabeal: R. Hobbs. Charlton) and the staff at the Pastoral and Veterinary Institute, Han&on, for use of their land. The authors express their appreciation to Mrs Carmel Young for conducting the survey and providing technical assistance, to Mr Stephen Drum and Mr Tim Mellington for technical assistance, to Mrs Arlene Daday and MS Sue Kleven for the serological analyses and to Mrs Libby Viccars for the PCR-based analysis of isolates. The research reported was supported by the Wheat Research Council and latterly, the Grains Research and Development Corporation.

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Amarger N. (1988) The microbial aspects of faba bean culture. In Nitrogen Fixationby Legumes in Mediterranean Agriculfure (D. P. Beck and L. A. Materon, Eds), pp. 173-178. Martinus Nijhoff, Dordrecht. Badaway N. S. (1982) Northern Wimmera Soils and Their Significance to Local Agriculture. Technical Report Series No. 54, Department of Agriculture, Victoria. Brockwell J. (1980) Experiments with crop and pasture legumes. In Methods for Evaluating Biological Nitrogen Fixation (F.J. Bergersen, Ed.), pp. 417488. Wiley Interscience, Chichester. Carter J. M., Gardner W. K. and Gibson A. H. (1994) Improved growth and yield of faba beans (Vicia faba cv. Fiord) by inoculation with strains of Rhizobium Ieguminosarum biovar. viciae in acid soils in southwest Victoria. Australian Journal of Agricultural Research 45, 613623.

Demezas D. H., Reardon T. B., Watson J. M. and Gibson A. H. (1991) Genetic diversity among Rhizobium leguminosarum bv. trtfolii strains revealed by allozyme and restriction fragment polymorphism analyses. Applied and Environmental

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Dudman W. F. (1977) S&olo&al methods and their application to dinitrogen-fixing organisms. In A Treatise on Dinitrogen

Fixation.

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(R.W.F. Hardy and A.H. Gibson, Eds), pp. 487-508. Gault R. R., Byrne P. T. and Brockwell J. (1973) Apparatus for surface sterilization of individual legume root nodules. Laboratory

Practice 22, 292-294.

Marshall D. J., Bateman J. D. and Brockwell J. (1993) Validation of a serial-dilution, plant-infection test for enumerating Rhizobium leguminosarum bv. viciae and its application for counting rhizobia in acid soils. Soil Biology & Biochemistry

25, 261-268.

Richardson A. E., Viccars L. A., Watson J. M. and Gibson A. H. (1995) Differentiation of Rhizobium strains usina the polymkrase’ chain reaction with random and dirt&d primers. Soil Biology & Biochemistry 27, 5 15-524. Silsbury J. H. (1991) Grain yield of Fiord faba bean in relation to strain of Rhizobium. Australian Journal of E.xperimental Agriculture 31, 251-254.