Pseudomonas cepacia, a potential suppressor of maize soil-borne diseases—Seed inoculation and maize root colonization

Soil Biol. Eiochem. Vol. 24, No. 10, pp. 999-1007, Printed in Great Britain. All rights reserved

0038-0717/92

1992

$5.00 + 0.00 Press Ltd

Copyright 0 1992 Pergamon

PSEUDOMONAS CEPACIA, A POTENTIAL SUPPRESSOR OF MAIZE SOIL-BORNE DISEASES-SEED INOCULATION AND MAIZE ROOT COLONIZATION K. P. HEBBAR,* A. G. DAVEY,~ J. MERRIN,~ T. J. MCLOUGHLINS Research

School

of Biological

Sciences,

P.O. Box 475, Canberra,

and P. J. DARTS

ACT 2601, Australia

(Accepted 25 April 1992) Summary-The effect of different inoculum densities and of varying methods of seed inoculation on the ability of the maize rhizobacterial strain Pseudomonas cepacia 526 (ATCC 53267) to colonize and multiply in the maize rhizosphere was studied in greenhouse and field trials. The amount of inoculum used had a significant effect on both the colonization and spread of P. cepacia on the roots and rhizosphere. However, even an inoculum as small as 30 bacteria seed-’ resulted in lo5 bacteria g-’ dry wt root after 2 weeks of plant growth in a montmorillonite clay vertisol soil from Darling Downs, Australia. After vigorous washing, root macerates still yielded P. cepacia indicating its close association to the roots. P. cepacia strain 64, which elicited high pectinase activity, had the capacity to penetrate maize root mucilage, while strains with low pectinase activity did not. The seed-inoculated bacteria spread rapidly to the newly-formed root surfaces of the seedling as well as on the adventitious (prop) roots in mature plants. The basal part of the root was colonized to a greater extent than the root tip region. Several field trials, conducted in the U.S.A. and in Australia using P. cepacia strains 526,406 (ATCC 53266) and 64, revealed that they can colonize maize roots under different pedo-climatic conditions and the type of inoculum (liquid or a peat-based) had no significant effect on their root colonization. The results indicated that for maize cultivars resistant to stalk rot, bacterial colonization of roots was highly variable, but this was not so for susceptible cultivars.

INTRODUCTION

to Suslow (1982) the initial population density of a bacterial inoculum is positively correlated with its ability to become established as a plant growth promoter on individual roots. Large populations of inocula in the immediate proximity of a germinating seed provide a competitive advantage over other soil microorganisms. However, plant growth promoting rhizobacteria (PGPR) differ in their ability to colonize and invade roots (O’Hara et al., 1981; Randhawa and Schaad, 1985). The average root populations of inoculant strains of PGPR in field trials with sugar beet ranged between 103-105cmm of root. In order to achieve uniform colonization and growth promotion the inoculum needed to contain at least 10’ colony forming units (cfu) per seed or 10’ cfu g-’ of dry inoculum (Suslow and Schroth, 1982). Ridge and Rovira (1968) reported that the spread of Azotobacter sp. from the point of inoculation was According

*Present address for correspondence: Centre de Pbdologie Biologique, UPR 6831 d; CNRS, associee B l’Univers% Nancv I. BP 5-54501, Vandoeuvre-les-Nancy, France. tPresent address: Wool Research and Development Corporation, 369 Royal Parade, Parkville, Victoria 3052, Australia. iPresent address: Department of Agriculture, University of Queensland, St Lucia, Qld 4072, Australia. @Present address: Stine Microbial Products, 4722 Pflaum Road, Madison, WI 53704, U.S.A.

restricted to the upper 2-3 cm of the root. The rate of movement of bacteria at a soil matric potential of 4.9 and 14.7 kPa was ranked as Pseudomonasjkorestens > Bacillus subtilis > Azotobacter vinelandii > Azotobacter chrococcum (Wong and Griffin, 1976). High migration rates are probably closely linked to high growth rates, both of which are important in primary colonization (Bowen and Rovira, 1976). For example, the generation time on seeds or roots was 5.2 h for Pseudomonas sp., 9-12 h for Rhizobium japonicum and 39 h for Bacillus sp. Therefore, rapid multiplication and migration of inoculants to newly-formed root surfaces from the point of inoculation may aid in their establishment and thereby protect the roots from pathogenic fungi. A comparative study of maize root-associated antagonistic bacteria revealed that P. cepucia strains were present in large numbers associated with the roots (Hebbar et al., 1992a) and when used as seed inoculum they were able to colonize maize roots and compete with the native soil microflora far better than other inoculated strains (Hebbar et al., 1992b). Production of antimicrobial agents, high growth rates, the capacity to utilize a wide range of carbon sources exuded by the roots and the production of extracellular enzymes were postulated to have provided the basis for their rapid colonization of maize roots. The aims of the present study deal with the ability of P. cepaciu strains to colonize the roots of maize 999

1000

K. P. HEBBAR ef al.

plants in both greenhouse and field trials and factors that might be important for seed bacterization. In particular, (a) the method of seed inoculation and quantity of seed inoculum, (b) the spread of the inoculum in the maize rhizosphere and its invasion into the root tissue, (c) the colonization and survival of inoculated bacteria in the rhizosphere and on the roots of maize seedlings (IO-14 days) and mature plants and (d) the effect of cultivar on root colonization were studied in detail. MATERIALS

AND METHODS

Maize cultivars

The cultivars used in greenhouse and field trials were GH5004, GH5010, GH5006 (supplied by Mr J. Colless, NSW Department of Agriculture, Agriculture Research and Advisory Station, Grafton, NSW 2460, Australia); XL94 (Dekalb Shand Seed Co., Goonoo Goonoo Road, P.O. Box 527, Tamworth, NSW 2340, Australia); JX2 1, JX180, ND 246, A654-900K, A654-310K, CO1109, A641 and J601 (Dr T. Burmood, Jacques Seeds Experimental Station, Prescott, Wisconsin, U.S.A.). Bacterial strains P. cepacia strains 526 (ATCC 53267) and 406 (ATCC 53266) were isolated from unsterilized root macerates of maize grown in Prescott soil (Madison, U.S.A.) whereas P. cepacia strain 64 was isolated from surface sterilized root macerate of maize grown at Kempsey (New South Wales, Australia) (Hebbar et al., 1992a). Spontaneous rifampicin-resistant (rif 50_100~gml-‘) mutants of the wild type bacteria were used as seed coatings in the colonization studies. These mutants were similar to the wild type strain in their phenotypic characteristics such as the colony morphology, ability to utilize sugars, their ability to suppress Fusarium moniltforme in vitro and colonize maize seedlings. Inoculum preparation and seed coating Broth inoculum. A loopful of a 24 h nutrient agar (NA, Difco) culture of P. cepacia were inoculated into 100 ml of nutrient broth (Difco) in 250ml Erlenmeyer flasks and incubated at 30°C in a shaker waterbath (250revmin’). A population of lo9 bacteria ml-’ had developed after 24 h of incubation. Seeds were coated with broth inoculum by dipping maize seeds for 10-15 min in a late log phase culture (lo9 bacteria ml-‘) or diluted nutrient broth cultures of P. cepacia. Dilution plating on rif NA plates was carried out to determine the populations of bacteria per seed. Peat inoculum. Peat inoculum was prepared by injecting 30ml of a late log-phase nutrient-broth culture (lo9 bacteria ml-‘) into 40 g of sterile y-irradiated peat in polyethylene packets. The packets were kept at 30-33°C for 10 days and then stored at 4°C. The P. cepacia populations in the peat carrier were

lo9 bacteria g-i, as determined by plating serial dilutions on nutrient agar. Seeds were coated with peat by mixing 1 g of peat inoculum with 1.5 g of 2% hydroxypropyl-5000sp sticker. Dilution plating on rif NA plates was carried out to determine the populations of bacteria per seed. Plant growth conditions in greenhouse

Darling Downs soil (pH 6.5, montmorillonite clay vertisol from Queensland, Australia) or a 40: 60 sand: Darling Downs soil mixture was used to study the effect of inoculum size of P. cepacia strains on their ability to colonize and spread along the roots and in the rhizosphere of lo-14 day old maize seedlings. Soft black plastic propagation tubes were filled with 700 g of soil or the sand: soil mixture and moistened with 100 ml of sterile water. The sand: soil mixture was used to facilitate its removal from the roots of seedlings and help minimize damage. Rhizosphere and root colonization of mature plants was tested in 25 cm dia pots filled with 7.6 kg of Darling Downs soil or 8.2 kg Hawkesbury soil (pH 6.6, silt loam from New South Wales, Australia). Seeds coated with bacteria were sown about 2cm below the soil surface. Three seeds were sown per pot and on germination thinned to one plant per pot. Plants were grown in a greenhouse, maintained at 25°C day and 15°C night temperatures, for IO-14 days or 2 months prior to the estimation of the bacterial inoculum populations from seedlings and mature plants respectively. After 2 weeks of plant growth the pots were watered daily and adjusted to field capacity. In ail the colonization experiments, uninoculated seeds were used as controls. Rhizosphere soil and root macerate dilutions from control plants were also plated on the selective rifampicin nutrient agar medium. Isolation of seed inoculants from rhizosphere and roots of maize Rhizosphere soil. In order to estimate the rhizosphere soil populations of inoculated bacteria, soil loosely attached to the roots was gently shaken off in 100ml of sterile Fahraeus N-free nutrient solution (Vincent, 1970) after which the serial dilutions were plated onto rif (50-1OOpg ml-‘) NA medium. Root wash. Following the procedure for estimating the rhizosphere microbial population, the residual bacterial populations on the root surface were estimated by washing the roots in 100ml of Fahraeus solution in a 250 ml Erlenmeyer flask containing 20 g of glass beads. The flasks were shaken in a rotary shaker at 250 rev min-’ for 20 min after which serial dilutions of root washings were plated as described above. Unsterilized root macerates. For estimating populations of bacterial inoculants adhering closely to the root surface, the roots after the above treatment were either washed in sterile N-free nutrient solution or, for field trials, were washed gently in running tap

Root colonization by P. cepucia water. Roots were then macerated in a Waring blender containing 100 ml of the nutrient solution for 3040 s. When small amounts (x 2-3 g f. wt) of roots were used, they were macerated in a motar and pestle to which lOm1 of nutrient solution was added. Surface-sterilized root macerates. For estimating populations of bacterial inoculants capable of invading the root tissue, washed roots were surface-sterilized in 3% hydrogen peroxide for 10 min followed by 45 washings in sterile water. Roots were then sterilized in 4% sodium hypochlorite for 15 min followed by several washings in sterile water to achieve complete surface sterilization. The efficacy of sterilization was tested by rolling the roots onto nutrient agar and found to be routinely effective. The surface-sterilized roots were macerated in sterile nutrient solution and serial dilutions were plated on agar medium. All plates were kept at 33°C for 48-72 h. Effect of seed inoculum level on the colonization and survival of P. cepacia strain 526 in maize rhizosphere and roots

Seed inoculation was performed by dipping unsterilized maize seedlings of cv. GH5010 for 15 min into a nutrient broth culture of P. cepacia strain 526 resistant to rifampicin and diluted to 2 x lo’, 2 x lo’, 2 x 10’ and 2 x 10’ cfu ml-‘. These dilutions resulted in populations of 7 x lo’, 7 x 103, 6 x 10’ and 3 x 10’ bacteria seed-’ as estimated by dilution plating. The population of bacteria on the seeds dipped in 2 x 1O’cfu ml-’ broth inoculum was higher than expected, possibly due to chemotaxis towards the seed coat. To obtain 10’ bacteria seed-’ with peat, a

inoculum containing 1O’cfu of strain 526 g-’ peat was used. The coated seeds were sown in 25 cm dia pots filled with moist Darling Downs soil. After 2 weeks of plant growth the population of seed-inoculated bacteria was estimated from the rhizosphere soil, root washings (bacteria adhering loosely to roots) and from unsterilized root macerates (bacteria adhering closely to the surface of the roots after root washings). The pots were not watered in this 2 week initial period to avoid washing the inoculum into the soil. Survival of the inoculum was verified further in the same experiment in a time course study where the bacterial populations were estimated from the rhizosphere soil and roots (root wash and unsterile root macerates) at 15, 30 and 60 days. Effect of inoculum size on the spread of P. cepacia On maize seedling roots. Maize seeds cv. GH5010 were dipped in late log-phase broth culture of P. cepacia strain 526 adjusted to 2 x lo’, 2 x lo’, 2 x 10’ or 2 x 10’ cfu ml-‘, and then sown in 40: 60 mixture of sand: Darling Downs soil in soft black plastic propagation tubes. The tubes were not watered after the seeds were sown. To check the extent of colonization on the roots of 10 day old maize seedlings, 5 cm pieces of basal, middle and tip regions of the primary root [Fig. l(a)] were macerated and plated on selective nutrient agar plus rifampicin medium. On mature maize roots. Maize seeds cv. JX180 dipped in a late log phase broth culture (lo9 cfu ml-‘) of P. cepacia strain 526 were sown in pots filled with

Darling Downs or Hawkesbury soils. After 2 months,

b 7 -:c:, BASE.*:.:.... -

1,-.

1001

i

BASE primary root

1,TIP

Fig. 1. Diagramatic representation of maize roots from (a) a maize seedling and (b) a mature plant. The thicker adventitious or prop roots emerge from the lower nodal regions of the stalk. The thinner median and inner adventitious roots emerge from the root collar region. The distance from the seed of basal, mid-root and root tip regions of maize seedling roots were ~0-5, ~7-12 and E 15-20 cm respectively.

1002

K. P.

HEEIBARet al.

the spread of the bacteria on to the thick nodal or prop roots and thinner adventitious roots located in the median or inner whorls of roots [Fig. l(b)] was determined in two separate experiments by plating root macerate dilutions on nurient agar plus rifampicin medium. Total bacterial populations were determined by plating root macerate dilutions on nutrient agar. Only one inoculum density (IO’ bacteria/seed) was used in this experiment. Invasion of maize roots by P. cepacia strains Maize seeds cv. GHS006 dipped in late log phase broth culture ( lo9 cfu ml-’ of P. cepacia strains 526 and 64 were sown in pots filled with Darling Downs soil. After 2 weeks of growth, the population of the inoculated strains adhering closely to the roots (from unsterilized root macerate) and within the root tissue (from surface-sterilized root macerates) was estimated by plating root macerate dilutions on rif NA media. The total populations of aerobic bacteria on the surface and within the root tissue were estimated on nutrient agar. Colonization of P. cepacia in field trials and effect of cultivar Field trials were conducted to test the efficacy of seed coating techniques in combination with a range of maize cultivars and soil types on the colonizing ability of P. cepacia strains. Several field trials were conducted in Australia (Grafton, an alluvial clay loam pH 5.3, New South Wales Department of Agriculture Research Station; and Kempsey, New South Wales, a silt loam pH 5.1) and in the U.S.A. (Sun Prairie, Prescott, Wise., a silt loam pH 7.1). The maize seeds were inoculated using either a peat inoculum as described previously or a liquid inoculum prepared by mixing peat inoculum with water as in trials conducted in Grafton (with cv. GH5004) and Kempsey (with cv. XL 94) or a nutrient broth inoculum (in Prescott trial, with cv. JX21). The liquid inocula were sprayed into furrows at sowing at the rate of ca lo8 bacteria 2.5 cm row-’ in Prescott trials and 10’ bacteria 6.5 cm row-’ in the Kempsey and Grafton trials. In a separate field trial conducted in Prescott, Wisconsin, the efficiency of seed inoculation was tested with maize cultivars that were either resistant (cv. ND246, A654-900K, A654-310K) or sensitive (cv. CO1109, A641 and 5601) to stalk rot. A Randomized Complete Block (RCB) with 4-5 replicate plots each of 9 x 4.5 m were used for field trials. After 2-3 months of plant growth, the roots were washed free of soil in running tap water, and were then macerated and plated on agar media as described above. Dilutions from root macerates and dilutions from uninoculated control plots were also plated on the selective rifampicin nutrient agar medium. Statistical analysis. The data from most of the colonization experiments were analysed using Genstat analysis of variance programme (Rothamsted _ Experimental Station, England).

RESULTS

Effect of seed inoculum density on the colonization and survival of P. cepacia strain 526 in maize rhizosphere and roots Colonization of maize seedlings. Significant differences (P < 0.01) existed in the bacterial populations of rhizosphere soil, root washings and root macerates of 10 day old maize seedlings, resulting from different amounts of inocula (Table 1). At inoculum densities of 7 x lo5 (log,, 5.8) bacteria seed-‘, the populations recovered from rhizosphere soil, root wash and root macerates were significantly larger than those arising from the other inoculum densities used (7 x lo3 to 3 x 10’ or log,,, 3.8 to 1.5 bacteria seed-‘). The decreases at lower inoculum densities were more evident in rhizosphere than in the root wash and root macerates. However, the difference in counts was much smaller at inoculum densities below 7 x 10’ bacteria seed-‘. At all inoculum densities, the rhizosphere soil counts were significantly smaller than the root macerate counts which in turn were lower than bacterial populations recovered from root washings. On an average 6.6 log,, cfu g-’ of P. cepacia were recovered from root macerates after vigorous washing of the roots with glass beads. Survival of P. cepacia strain 526. The effect of inoculum density on the populations of P. cepaciu strain 526 in rhizosphere soil, root wash and root macerates was examined 15,30 and 60 days after seed inoculation (Fig. 2). Inoculum densities significantly (P < 0.01) affected the populations of P. cepacia in the rhizosphere and on roots of 15 day old plants. The inoculation of lo5 bacteria seed-’ using a broth inoculum resulted in the largest populations, followed by inoculation at 10’ bacteria seed-’ with peat and IO3 and 10’ bacteria seed-’ in broth inoculum. Recovery of inoculum from both the seedling and mature plants indicated that after 15 days of plant growth, the maximum population or the “colonization potential”, was influenced by initial inoculum

Table I. Effect of inoculum level of P. cepacio strain 526 on its colonization and survival on roots and in rhizosphere soil of 2 week old maize* grown in Darling Downs soil Location Inoculum level/ saedt (log,,) 7 7 6 3

x x x x

IO5 (5.8) lO’(3.8) IO’ (1.8) IO’ (1.5)

Mean SEDg Significant

Rhizosphere Root soil wash Countsf (log,, cfu g-

’

Root macerate dry wt)

6.2 5.1 4.1 3.4

8.5 7.2 6.8 6.5

7.8 6.4 6.2 5.9

4.7

7.3 0.25 0.01

6.6

P

Mean 7.5 6.2 5.7 5.3 0.29 0.01

‘Maize cv. GH5010. Weeds were inoculated by dipping in a nutrient broth culture of rifampicin resistant P. cepacia adjusted to IO’, IO’, IO’ and 10’ bacteria ml-‘. $Mean of 3 replicate plants from which serial dilutions of samples were plated on NA + rifampicin plates. ^ ..^^ . .

Q>tanCtara

error

Of dltterences.

Root

*r

Root

colonization

macerate

6

Rhizosphere

15

30

soil

60

Plant growth days Fig. 2. Effect of inoculum level and plant age on the colonization and survival of P. cepacia strain 526 in the rhizosphere soil and on roots of maize. Values are the means of 4 replicate plants. Effect of inoculum level and type [(m) 10s peat; (0) 10’ broth; (x) lo3 broth; (0) 10’ broth] on root colonization and survival was highly significant up to 15 days of plant growth. SED = standard error of differences of means

(P <<0.0 1).

density in the range lo’-10’ bacteria seed-‘. However, this effect was less evident after 30 days of plant growth. After 30 days, seed treated with broth at IO’cfu seed-’ again developed the largest root populations. However, there were no differences in the populations resulting from peat inoculation at 10s bacteria seed-’ or the lo3 and 10’ broth inoculation treatments. After 60 days the smallest inoculum density (10’ bacteria seed-‘) resulted in significantly smaller root populations (Fig. 2). However, no significant difference was evident in bacterial populations using other inoculum densities. Root wash populations were significantly larger than root macerate and rhizosphere soil populations colonizing mature plants (Fig. 2), a similar result to that obtained from seedlings (Table 1). Efect

on inoculum level on the spread of P. cepacia

On the primary root of a maize seedling. The extent of colonization of P. cepacia strain 526 on the roots

by P. cepacia

1003

of a 10 day old maize seedling grown in a sand: soil mixture showed that the primary root [Fig. l(a)] was colonized moderately well at all densities of inoculum (Table 2). However, root populations of P. cepacia were highest following inoculation with lo5 bacteria seed-‘. No significant differences existed between populations developing from inoculum densities of lo3 to 10’ bacteria seed-‘. The results indicated that an inoculum of lo5 bacteria seed-’ was necessary to ensure a uniform colonization along the whole root. The population of P. cepacia colonizing the basal (x:0-5 cm from the seed) or oldest region of the primary root was significantly larger than on the mid-root (~7-12 cm from the seed) or root tip regions (Z 15-20cm from the seed). Furthermore, movement of the inoculum from its initial location on the seed had kept pace with the root development, covering a distance of >20 cm in 10 days. On mature roots. As a maize plant matures, growth of the primary root is arrested and adventitious (nodal or prop) roots develop from lower nodes of the stalk and from the crown region (median and inner roots) [Fig. l(b)]. In two separate experiments, spread of seed-inoculated P. cepacia strain 526 on the prop, median and inner roots was compared. Significant difference occurred in the spread of P. cepacia between the root regions (P < 0.025) and also in relation to the soil type (P -C0.01) [Table 3(a)]. When the plants were grown in Hawkesbury soil, the population of P. cepacia was greater on the older basal regions of both prop and median roots than on other parts of the root. However, in Darling Downs soil all regions of the root were equally well colonized by P. cepacia. Furthermore, the results revealed that the pattern of colonization of roots by native soil bacteria also varied with root region. The root tip region was colonized by a larger population of soil bacteria than the thicker mid or basal region [Table 3(b)]. There were no significant differences in the populations of seed-inoculated P. cepacia reisolated from prop, median and inner roots recovered from Darling Downs soil [Tables 3(a) and 41. However, Table 2. Effect of inoculum level on the spread and recovery of P. cepacia strain 526 from different regions of the primary roots of 10 day old maize* grow” in Darling Downs soil Location on the root? Inoculum level/ seed1 (log,,) 7x 7x 6x 3x

105(5.8) 10) (3.8) IO’ (1.8) IO’ (1.5)

Mea” SED Significant

Tip Base Mid Counts5 (log,, cfu per 5 cm root-‘) 3.9 4.1 4.7 4.4

4.9 2.8 3.4 3.1

4.5 2.5 3.0 2.9

4.7

3.6 0.25

3.2

0.01

P

Mean 5.1 3.1 3.7 3.5 0.29 0.01

*Maize cv. GH5010. t&e Fig. l(a). $Seeds were inoculated by dipping in a nutrient broth culture of rifampicin resistant P. cepacia adjusted to IO’, IO’, IO’ and IO1 bacteria

ml- ‘.

$Mean of 3 replicate plants from which serial dilutions of 5 cm pieces of root macerates were plated on NA + rifampicin plates.

K. P.

1004

HEBBARet al.

Table 3(a). Spread and recovery of seed-inoculated P. cepacia strain 526 from different root regions and root types of 2 month old maize* plants Root type Prop root Median root Counts~ (log,, cfu g-’ root)

Soil

Location?

Hawkesbury pH 6.5

Base Mid Tip Root mean Significant P

5.0 4.3 4.4 4.5

Darling Downs pH 6.6

Base Mid Tip Root mean Significant P

4.7 5.2 5.3 5.1

Effect of root types, location and soils

Root mean SED Significant P

4.8

Location mean

Soil mean

4.9 4.3 3.9 4.3

4.9 4.3 4.1

4.4

5.4 5.5 5.1 5.3

5.0 5.3 5.2

5.2

0.265 0.025

0.15 0.01

NS

NS 4.8 0.15 NS

‘Maize cv. JX180 seeds coated with rifampicin-resistant P. cepacia 526 and grown in Hawkesbury (silt loam) or Darling Downs soil (vertisol) and the spreading of the bacteria on to the prop and median adventitious roots were determined by plating root macerate dilutions on NA + rifampicin plates. t5 cm long root pieces from different regions [see Fig. l(b)] were used for isolation. ZMean of 4 replicate plants.

Table 3(b). Total bacterial populations on different root types and root regions of 2 month old maize’ plants inoculated with P. cepacia strain 526 Root type Prop root Median root Countsi (log,. cfu p.-’ root)

Soil

Locationt

Hawkesbury

Base Mid Tip Root mean SED Significant P

7.3 1.6 8.4 7.7

Base Mid Tip Root mean SED Significant P

7.8 8.4 8.8 8.3

Root mean SED Significant P

8.0

Darling

Downs

Effect of root types, location and soils

Location mean

Soil mean

7.5 8.4 8.6 8.2

7.4 8.0 8.5

8.0

8.2 8.6 8.9 8.6

8.0 8.5 8.8

8.4

0.11 0.01

0.09 0.01

0.11 0.01

0.11 0.01 8.4 0.09 0.01

*Maize cv. JX180 seeds were coated with strain P. cepacia 526 and grown in Hawkeshury or Darling Downs soil; total bacterial populations on the prop and median adventitious roots were determined by plating root macerate dilutions on NA plates. t5 cm long segments from each region of the root were used for isolation [see Fig. I(b)]. iMean of 4 replicate plants.

roots recovered from Hawkesbury soil showed significantly higher populations of P. cepacia on the inner roots than on the prop or the median roots (Table 4). On the contrary, the total bacterial populations g-’ dry wt root from the inner, median and prop roots differed significantly irrespective of the soil from which they were recovered [Tables 3(b) and 41. The populations adhering to the roots from Hawkesbury soil were in the order Inner roots > Median roots > Prop roots and in Darling Downs soil it was Inner roots = Median roots > Prop roots. Invasion of maize roots by P. cepacia strains

Studies on the colonization of maize roots by seed-inoculated P. cepacia strains revealed that differences existed in their ability to “invade” maize roots (Table 5). P. cepacia strain 64 was recovered from

surface sterilized root tissue at lo3 bacteria gg’ dry wt root. However, strain 526 could not be isolated from macerates of surface-sterilized roots indicating that strain 64 formed closer associations with the maize roots than strain 526. Strain 64 accounted for 78% of the population recovered following surface sterilization. P. cepacia strains 526 and 64 comprised 83-88% of the total populations from unsterilized root macerates. The size of the total bacterial populations recovered from both unsterilized and surfacesterilized root macerates of P. cepacia treated seedlings were similar to those recovered from the uninoculated control plants. Root colonization by P. cepacia strains infield trials

The multi-locational trial showed that each of the three strains 526, 406 and 64 were recovered from

Root colonization Table 4. Effect of soil and maize* root type on the recoveryt of population of P. cepacia strain 526 and total bacteria

by P. cepacia

1005

Table 6. Survival of P. cepacia strains on maize roots in field trials* Location*

Root type Prop Inner root root Counts$ (log,, cfu g-’ root)

Bacteria soil 526 526

Hawkesbury Darling Downs Root mea” Significant P

5.5 5.2 5.3

4.5 5.5 5.0 NS

Interaction soil-root type SED Significant P Hawkesbury Darling Downs Root mean SED Significant P

5.0 5.3 5.2 NS

0.351 0.025 11.8%

CY.

Total Total

Soil mea”

7.1 8.1 1.9

8.6 8.5 8.5

8.2 8.3 8.2

0.123 0.01

Interaction soil-root type SED Significant P

NS

0.175 0.1 3.1%

C”.

*Maize cv. JX180 seeds coated with P. cepacia strain 526 and grow” in Hawkesbury and Darling Downs soil for 2 months. tRecovery of inoculurn from prop and inner adventitious roots was determined by plating root macerate dilutions on NA + rifampicin plates whereas NA was used for estimating total bacteria. IValues are the mean of 6 replicate plants.

Resistant

q Susceptible

SED

10.119

Strain

Inoculumt

Madison Grafton Kempsey (PH 5.3) (PH 5.1) (PH 7.1) Counts1 (cfu g-’ fresh wt root + SE)

526

Peat Liquid Peat Liquid Peat Liquid

4.7 f 5.1 f 5.8 f 5.8 k 5.1 f 5.3 f

64 406

0.98 0.86 0.64 0.78 0.53 0.58

4.5 f 0.37 4.5 + 0.28 5.4 f 0.47 5.3 + 0.49 ND 4.9 * 0.50

ND 4.6 + 0.60 ND ND ND ND

*Field trials were conducted at Grafton (alluvial clay loam) and Kempsey (silt loam) in Australia and in Prescott (silt loam), Wis., U.S.A. tBoth peat-based and liquid inocula of P. cepacia strains 526 and 406 isolated originally from Madison (Prescott) and strain 64 isolated from Kempsey soil were used for coating seeds. IMean f SE of l&20 replicate plants sampled after 2 months of planting at Kempsey and 3 months at Grafton and Madison. ND not determined.

roots of 2 to 3 month old plants at densities of 10s bacteria g-’ fresh wt root (Table 6). However, populations of P. cepaciu strain 64, a Kempsey isolate, were consistently higher than those of strains 526 and 406 in both Kempsey and Grafton soils. The strains were recovered in the order: 64 > 406 > 526. Irrespective of whether peat or liquid inocula were used, no effect on the colonization of roots by P. cepacia in both Grafton and Kempsey trials was detected. The effect of maize cultivar on colonization in the field was highly significant (P < 0.01) (Fig. 3). For the cultivars resistant to Fusarium stalk rot, colonization was highly variable, but between susceptible cultivars, differences were not significant. However, the population was, on average, above lo4 bacteria g-’ fresh wt root in most of the cultivars tested. DISCUSSION

Cultivars Fig. 3. Effect of cultivar on the colonization of maize roots by P. cepucia strain 526 in the field. Maize cultivars ND246, A654-900K and A654-310K are resistant to stalk rot; cultivars A641, CO1109 and 5601 are susceptible to stalk rot. Bacterial counts are means of 4 replicate plots sampled after 2 months of plant growth. Effect of cultivar on colonization significant. SED (P < 0.01).

Bowen and Rovira (1976) reported that rapid migration rates of microbial inoculants on roots are probably linked to high growth rates. Consequently the beneficial effect, anti-fungal activity in this case, of the bacterial inoculum will not be confined to the seed coat, but will also occur in the rhizosphere and on the roots as the inoculum migrates along with the growing root. The results of the present study indicated that the initial size of the inoculum of P. cepacia

Table 5. Ability of P. cepacia strains to invade maize’ roots Root treatment

Strain 526

Populationt P. repack2

Total bacteria 64 Uninoculated

P. cepacia

Total bacteria 526 or 64 Total bacteria

Unsterilized root Sterilized root macerate macerate Countsf (log,, cfu gg’ dry wt root + SE) 7.4 f 8.4 k 7.2 f 8.6 f 0 8.4 +

0.40 0.18 0.31 0.24 0.22

0 4.1 k 3.5 f 4.2 f 0 4.7 +

0.32 0.76 0.60 0.28

*Maize cv. GH5006. tunsterilized and surface sterilized root macerates were plated on nutrient agar for estimating total bacterial populations and on nutrient agar plus rifampicin for estimating the populations of P. cepacia strains 526 and 64. SValues are the means of 5 replicates f SE (standard error).

1006

K. P.

HEBBAR

had some effect on its maximum population attained, the “colonization potential” on maize seedlings. However, this effect was less evident in mature plants due to the ability of the bacteria to colonize the rhizosphere and roots of maize rapidly. Bennet and Lynch (1981), who studied the effect of inoculum density on growth and steady-state populations of Pseudomonas sp., Mycoplasma sp., and a Curtobacterium sp. in the rhizosphere of an axenically-grown barley plant reported that the colonization potential was not influenced by the inoculum density. For example, inoculation of barley seedlings at concentrations of 1 x lo’, 1 x lo5 or 1 x 10’ viable cells mg-’ dry wt root resulted in rapid root colonization with a maximum population of 5 x 10’ viable cells mg-’ dry wt root being attained at all inoculum concentrations. However, in greenhouse experiments under non-sterile conditions the colonization potential of Arthrobacter crystallopoietes in the rhizosphere of barley was dependent upon the initial inoculum density (Kirillova et al., 1981). Also, it was demonstrated that the colonization of barley roots by Rhizobium leguminosarum increased with the higher initial inoculum densities for up to 13 days following inoculation, but it later stabilized to 103-lo4 bacteria cm-’ of root. Similar results were also obtained in our study. The capacity of P. cepacia strain 526 to migrate rapidly to newly-formed root surfaces of maize seedlings and also on the adventitious (prop) roots of mature plants, from the point of inoculation (i.e. the seed) is an important characteristic for a biocontrol strain which antagonizes fungal growth. Establishment of the bacteria on these later formed roots and mature roots may protect them from “late infecting” pathogens [Tables 3(a) and 41. The basal region of mature maize roots is thicker than the mid or root tip regions, as a consequence of which the surface area is larger. Hence, comparisons of bacterial attachment to root pieces with different diameters may be confounded by this factor as surface area tends to increase linearly and root weight geometrically with a corresponding increase in root diameter. However, differences in the ability of P. cepacia strain 526 to colonize the basal, mid or root tip region (on root weight basis) was larger than could be accounted for by surface area alone, indicating intrinsic differences in colonization potential between various portions of the root system. This difference was evident in maize roots growing in Hawkesbury soil than those growing in Darling Downs soil [Table 3(a)]. Larger numbers of soil bacteria were isolated from the root tip region than from the thicker mid or basal region, which in turn supported smaller numbers of P. cepacia [Table 3(a) and (b)]. This may be due to the younger root tip region being able to support a more competitive community, resulting therefore in a smaller population of the inoculum. This may be also due to the inability of the inoculum to grow with the root tip. Variation in the colonization of different types of

et

al.

roots by rhizosphere microorganisms has been reported by Sivasithamparam and Parker (1979) who compared the microbial communities colonizing both seminal and nodal roots of wheat. It was found that thinner seminal roots supported significantly higher numbers of bacteria, actinomycetes and fungi in their rhizosphere than did the thick nodal roots. In the present study, although the thinner inner roots of maize also supported a larger total bacterial community than the thicker prop roots (Table 4) this did not significantly affect the population of seed-inoculated P. cepacia, on the contrary, in Hawkesbury soil they were higher in the inner roots (Table 4). Therefore colonization potential of a bacterial strain may not only be influenced by the root system but also by the soil type in which it is tested. Even after vigorous washing with glass beads, P. cepacia was still recovered from maize root macerates indicating its close attachment to maize roots. The ability of antagonists such as P. cepaciu strain 64 to attach closely to or “invade” maize roots may play an important role in its ability to protect the plant from fungal pathogens by limiting their spread through the root tissue. It was reported, for instance that Fusarium moniliforme penetrates root tissue as a first step towards infection of roots and the stalks (Voorhees, 1934; Lawrence et al., 1981). Surface sterilization of roots is another means by which the efficacy of bacterial invasion and attachment can be assessed. The use of triphenyl tetrazolium chloride (TTC) prior to and following surface sterilization of maize seedling roots has been used by us to assess the depth to which the tissue is killed by surface sterilants. Hydrogenation of TTC forms a red, stable and non-diffusible triphenyl formazan indicating viable cells and the dead cells remain colourless (Parkinson et al., 1971). In the present study, to achieve complete surface sterilization, indicated by agar roll tests, the roots had to be sterilized for 10 min in H,02 followed by 15 min in hypochlorite, even though complete root tissue death occurred after only 5 min sterilization in hypochlorite alone (unpublished data). Survival of P. cepacia strain 64 suggested that although the root tissue cells were killed, the thick mucilagenous layer covering the roots may have protected the inoculant from the action of the surface sterilants, whereas strain 526 with its consequent inability to penetrate the mucilagenous layer was unprotected (Table 5). Scanning electron micrographs and transmission EM sections of roots infected with strain 64 also showed that this strain was located just beneath the mucilagenous layer covering the cell wall rather than within the root cortex (K. P. Hebbar, unpublished Ph.D. thesis, Australian National University, 1986). The ability of P. cepacia strain 64 to colonize maize roots in field trials conducted in Grafton and Kempsey in significantly larger numbers than strains 526 and 406, may also reflect its closer root association (Table 6). Cultivar differences affect both fungal (Andal et al., 1956; Buxton, 1957) and bacterial (Rennie and

Root colonization by P. cepucia

Larson, 1981) colonization of plant roots. Rennie and Larson (198 1) reported increased associative nitrogenase activity in the rhizosphere of spring wheat lines where there was a substitution of chromosomes involved in root rot resistance. Root rot susceptible lines supported larger numbers of rhizosphere bacteria, especially a N,-fixing Bucilius polymyxa, presumably because of greater root exudation. Although, the present study has also shown the colonization was variable between cultivars, the cultivars sensitive to stalk rot did not support a higher population of the P. cepacia strain 526 than the resistant cultivars (Fig. 3). In a study of the effects of various N,-fixing bacteria on the nitrogen contents of four different maize hybrids, Saric et al. (1987) concluded that the formation of positive root-associations depended on the specificity of N,-fixing bacteria to the corn hybrids. The effect of bacterial inoculation on the nitrogen concentration in the hybrids ranged from highly positive to highly negative. The extensive use of rhizobacteria for the biological control of soil-borne fungal diseases is usually restricted by their ability to control only a particular plant disease or by their low capacity to survive in a specific soil type (Papavizas, 1985). Strains of P. cepacia with broad spectrum anti-fungal activity have been reported as potential plant growth promoting rhizobacteria (PGPR) of lettuce (Homma et al., 1989), peas (Parke, 1990) and sunflower (Hebbar et al., 1991). Results of the present study revealed that P. cepacia strains have the ability to colonize roots of maize growing under different pedo-climatic conditions irrespective of the soil pH or soil type and are therefore potential suppressors of maize soil-borne diseases. Biocontrol assays in the presence of maize fungal pathogens will be needed to confirm these findings. Acknowledgements-=The

authors gratefully acknowledge Mrs Barbara Setchell for her excellent technical assistance and Agrigenetics Research Corporation, Madison, Wis., U.S.A., for funding the project. We also thank Drs 0. Berge and T. Heulin for their valuable comments,

REFERENCE

Andal R., Bhuvaneswari K. and Subba Rao N. S. (19.56) Root exudates of paddy. Nature 178, 1063. Bennet R. A. and Lynch J. M. (1981) Colonization potential of bacteria in the rhizosphere. Current Microbiology 6, 137-138.

Bowen G. D. and Rovira A. D. (1976) Microbial colonization of plant roots. Annual Review of Phy~opathoio~y 14, 121-144.

Buxton E. W. (1957) Some effects of pea root exudates on physioio~~l races of Fusurium oxysparum Fr. f. pisi (Lini). Transactions of the British ~y~o~ogica~ Society 40, 145-154.

Hebbar K. P,, Davey A. G. and Dart P. J. (1992a) Rhizobacteria of maize antagonistic to Fasarium moni~l~orme, a

1007

soil-borne fungal pathogen: isolation and identification. Soil Biology & Biochemistry 24, 979-987.

Hebbar P., Berge O., Heulin T. and Singh S. P. (1991) Bacterial antagonists of sunflower (Helianfkus annuus L.) fungai pathogens. Pfanr and Soil 133, 131-140. Hebbar K. P., Davey A. G., Merrin J. and Dart P. J. (1992b) Rhizobacte~a of maize ~tagonistic to Fusarium moniliforme, soil-borne fungai pathogen: colonization of rhizosphere and roots. Soil Biology & Biochemistry 24, 989-997.

Homma Y., Sato Z., Hirayama F., Konna K., Shirahama H. and Suzuki T. (1989) Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soilborne plant pathogens. Soil Biology & Biochemistry 21, 723-728.

Kirillova N. P., Stasevich G. A., Kozhevin P. A. and Zvyangintsev D. G. (198 1) Bacterial population dynamics in a soil-plant system. Microbiologiyn 50, 128-133. Lawrence E. B., Nelson P. E. and Ayers J. E. (1981) Histopathoiogy of sweet corn seed and piants infected with F~ar~urn moniil~orme and F. oxysporam. Pkyto~rho~ogy 71, 379-386. O’Hara G. W., Davey M. R. and Lucas J. A. (1981) Effect of inoculation of Zea mays with Azospirillum brasilense strains under temperate conditions. Canadian Journal of A4icrobioiogy 27, 871-877.

Parke J. L. (1990) Efficacy of Pseudomon~ cepaciu strain AMMD and Pse~domonas~uorescens strain PRA25 in biocontrol of Pytkium damping-off and Apkunomyces root rot of peas. In Plant Growth-Promoting Rhizobacteria-Progress and Prospects (C. Keel, B. Kolier and G. Difago, Eds), p. 30. Proceedings of The Second International Workshop on Plant Growth Promoting Rhizobacteria, Interlaken. Parkinson D., Gray T. R. G. and Williams S. T. (1971) ~et~o~~or Studying the Ecology of Soil microorganisms. Blackwell, Oxford. Papavizas G. C. (1985) Trickoderma and Giiociadum: biology, ecology and potential for biocontrol. Annual Review

ofPkytopatkology 23, 23-54.

Rennie R. J. and Larson R. I. (1981) Dinitrogen fixation associated with disomic chromosome substitution lines of spring wheat in phytotron and in the field. In Associative ~=~~at~on (P. B. Vose and A. P. Ruschel, Eds), Vol. I, pp. i45-154. CRC Press, Boca Raton. Rhandawa P. S. and Schaad N. W. (1985) A seedling bioassay chamber for determining bacterial colonization and antagonism on plant roots. Pkytopathology 75, 254-259.

Ridge E. H. and Rovira A. D. (1968) Microbial inoculation of wheat. Transactions 9rh ~nIerna~iona[ Congress of Soil Science,Adelaide 3, 473-481.

Saric M. R., Saric 2. and Govedarica M. (1987) Specific relations between some strains of diazotrophs and corn hybrids. Plant and Soil 99, 147-162. Sivasithamparam K, and Parker C. A. (1979) Rhizosphere microorganisms of seminal and nodal roots of &heat grown in pots. Soil Biology & ~~ochemisfry 11, 155-160. Sustow T. W. (1982) Role of root-colonizing bacteria in plant growth. In Phytopatkogenic Prokaryofes, Voi 1, Chapter 8, pp. 187-223. Academic Press, New York. Suslow T. V. and Schroth M. N. (1982) Rhizobacteria of sugar beet: effects of seed applications and root colonization on yield. Pkyropatkology 72, 199-206. Vincent J. M. (1970) In A Manualfor the Practical Study of Root Nodule Bacteria. IBP Handbook No. 15. Biackweli, Oxford. Voorhees R. K. C. (i934) Histological studies of a seedling disease of corn caused by Gibberella moniiiforme. Journal of Agricultural Research 49, 1009-1015.

Wong P. T. W. and Griffin D. M. (1976) Bacterial movement at high matric potential I. In artificial and natural soils. Soil Biology & Bjockemjstry 8, 215-218.