Potato Cultivar, Pathogen Isolate and Antagonist ... - PubAg - USDA

Biocontrol Science and Technology (2000) 10, 267-279

Potato Cultivar, Pathogen Isolate and Antagonist Cultivation Medium Influence the Efficacy and Ranking of Bacterial Antagonists of Fusarium Dry Rot D. A. SCHISLER,! P. J. SLININGER,1 L. E. HANSON 2

AND

R. LORIA 2

1 National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, IL 61604, USA; 2 Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA

(Received/or publication 6 August 1999; revised manuscript accepted 6 January 2000)

The process of selecting biological control agents for further development frequently does not involve conducting bioassays of strain effectiveness on a range of pathogen isolates or host cultivaI's. Additionally, though previous studies have demonstrated that the medium used to produce biomass ofan antagonist can alter its efficacy, this factor is also rarely considered when selecting for the most effective antagonist. Host cultivaI', pathogen isolate, and the cultivation medium used to produce the antagonists' biomass were examined as factors ofpotential importance for assessing the relative effectiveness of bacterial biocontrol strains accurately. Five bacterial antagonists that control Fusarium dry rot on stored potato tubers were assayed for effectiveness against 10 isolates of Gibberella pUlicaris. All antagonists reduced disease severity (35-81%) regardless of the specific assays conducted. Howevel~ when the antagonists' biomass were produced on two media that differed both in nutrient composition and phase, the efficacy ranking of antagonist Enterobacter sp. S11:P:08 varied from first to fourth most effective. For the antagonists studied, the phase of a nutritionally identical medium had little impact on the efficacy ranking of the five antagonists. Four of the five antagonists had efficacy rankings that ranged from first to last depending on the isolate of the pathogen used to conduct the bioassay. The cultivar of the host also caused variations in the efficacy ranking of the antagonists. These results indicate that bioassays should be conducted using a range of liquid culture production media, pathogen isolates, and host cultivars in order to choose an antagonist that has the highest likelihood for commercial development as an effective biological control product.

Keywords: microbial selection strategies, biological control, biocontrol, Fusarium dl)' rot, Gibberella pulicaris, Fusarium sambucinum

Correspondence to D. A. Schisler. Tel: + 13096816284; Fax: + 13096816427: E-mail: schislda@ mail.ncaur. usda.gov Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. ISSN 0958-3157 (print)/ISSN 1360-0478 (online)/00/030267-13

© 2000 US Government

268

D. A. SCHISLER ET AL.

INTRODUCTION Over the past few decades, a sharp increase in research activity directed towards biological control of plant diseases (Baker, 1987) has resulted in only a comparatively modest development of commercially available products (Fravel & Larkin, 1996). The factors that account for the relative scarcity of commercially available biocontrol products include but are not limited to: unfavourable economics of biomass production or market size, biomass stabilization or formulation difficulties, inadequate patent protection of potential products, inconsistent product performance, and registration difficulties or expenses. In recent years, considerable progress has been made in the application of increasingly sophisticated molecular tools and ecological theory from related biology subdisciplines to address some of the product performance issues (Kinkel, 1997; Pierson et al., 1998: Duffy & Defago, 1999). These advances should result in more consistent reduction of disease when using biological control products as knowledge of the physiology and ecology of the antagonist, target pathogen, host plant, and associated microflora increases. Another factor that contributes to the scarcity of biocontrol products is that researchers rarely consider the importance of liquid production media, pathogen isolate, and host cultivar on the efficacy of biocontrol agents when they select antagonists for further development (Cook, 1993). As a result, strains that are unsuitable for commercialization or unlikely to be effective against a wide range of pathogen isolates and host environments can be mistakenly chosen as the 'best' strains. Laboratory biocontrol agents are often chosen for additional research without considering the realities of mass producing the strains for commercial development (Knudsen et al., 1997). For yeast or bacterial strains, commercial-scale production of biomass will require liquid culture production in industrial scale fermentation vessels. The biocontrol efficacy of microbial strains can vary greatly depending on the medium used to produce the biomass and strains can differ radically in their amenability to production in liquid culture (Slininger et al., 1994; Schisler & Slininger, 1997). Strains that perform well when grown on solidified media, a common production practice in laboratory bioassays, can possess inadequate or inferior liquid culture growth kinetics (Slininger et al., 1994). In previous work, we used liquid culture growth kinetics and the biocontrol efficacy of strains that were produced in liquid culture to rank the commercial development potential of bacterial strains that demonstrated excellent efficacy in preliminary bioassays, in which biomass was produced on a solid medium. However, little is known regarding whether the phase or nutrient content of the antagonist cultivation medium are also important variables to consider in selecting strains that have a high potential for commercial development. Furthermore, although there are reports on the influence of host cultivar on the colonization of the host by a biocontrol agent and the degree of biocontrol exerted (King & Parke, 1993; King & Parke, 1996), there have been no studies, to our knowledge, that have determined if host cultivar influences the efficacy ranking of a group of biocontrol strains. Similarly, although it is known that a biological control agent may be more effective against one pathogen strain than another (Schisler & Slininger, 1994), it is not known how the choice of a pathogen strain for a biocontrol bioassay would influence the efficacy ranking of biocontrol strains. The development of a biological control product is ideal for the control of Fusarium dry rot of stored potato (Solanum tuberosum L.) (Schisler et al., 2000). Using biological control of Fusarium dry rot as a model system in the present studies, we evaluated the influence of host cultivar, pathogen isolate, and the cultivation medium used to produce a biological control agent on the efficacy and ranking of five bacterial antagonists of Fusarium dry rot of stored potato tubers. The objective of the studies was to determine if any, or all of these factors, should be evaluated to select candidate biocontrol strains accurately according to their actual potential for effectiveness when developed and deployed as commercial biological control products.

EFFICACY RANKING OF BIOCONTROL AGENTS

TABLE I.

269

Antagonist strain designation and identification of bacterial antagonists that reduce the severity of Fusarium dry rot disease NRRL Accession No."

Antagonist

B-21128 B-21132 B-21133 B-21134 B-21050

S09:Y:08 SII:P:08 SI1:P:12 SII:P:14 SII:T:07

Identification' Pseudomonas jfuorescens bv. I' Enterobacler sp. P.fiuorescens bv. V P.jfuorescens bv. V Enterobacler cloacae'

aNRRL Patent Culture Collection. National Center for Agricultural Utilization Research. Peoria. IL. USA. 'Bacterial strains preliminarily identified by Biolog GN microplates and gas-chromatographic analysis of phospholipid fatty acids. Presumptive identifications confirmed by conducting published biochemical and physiological tests of taxonomic utility as previously described (Schisler & Slininger. 1994). 'Identification confirmed by the American Type Culture Collection. Rockville. MD. USA.

MATERIALS AND METHODS Common Methods for Bioassays of Bacterial Antagonists Against FusariullI Dry Rot

Five strains of Gram negative bacteria (Table I) were utilized in all studies. Previous studies have demonstrated the effectiveness of these strains in reducing the severity of Fusarium dry rot of potato tubers (Schisler & Slininger, 1994; Schisler & Slininger, 1997). These five strains were the best of 18 strains, based on their superior growth kinetics and efficacy when produced in liquid culture (Slininger et al., 1994). All bacterial strains were originally isolated from periderm enriched soils that were suppressive to the development of Fusarium dry rot (Schisler & Slininger, 1994). Ten isolates of Gibberella pulicaris were used individually in biological control bioassays and were isolated from seed, tablestock, and processing tubers obtained from various sites in the Northeastern United States (Table 2; Hanson et al., 1996). Tubers used in bioassays were obtained from commercial suppliers of seed potatoes or commercial retail markets. Methods for conducting bioassays on the biological control of dry rot have been reported previously (Schisler et al., 1997). Briefly, potato tubers were stored at 5-7°C. Tubers were washed in deionized water and warmed to 20°C 24 h prior to use. Conidial inoculum of G. pulicaris was produced in Petri dishes of clarified V-8 juice agar (CV8 agar) under 12 h/day fluorescent light for 7 days at 24°C (Schisler & Slininger, 1994). Suspensions of macroconidia TABLE 2.

Origin of G. pulicaris isolates used

Isolate number 15-3/1 34-4(1

41-1" AICaND5" KaAFlla LaMc2' NoHe5 v NoT&S4 V SJl84Cv SJ561Bv

Tuber origin

US State origin New York (NY) NY NY NY Pennsylvania (PA) NY PA PA Maine (ME) ME

a.v Bacterial antagonists were tested against G. pulicaris groups A and 'B'.

Seed Seed Seed Seed Tablestock Seed Processing Processing Seed Processing In

separate experiments

270


were obtained by flooding the surface of colonized CV8 agar with cold (4°C), weak P0 4 buffer (0.004% phosphate buffer (pH 7.2) with 0.019% MgCl z ; Aid-Pack USA, Gloucester, MA, USA) and dislodging conidia using a sterile inoculating loop. Two or three days before use, strains of bacterial antagonists were recovered from storage in 10% glycerol at - 80°C by streaking partially thawed glycerol onto 115 strength tryptic soy broth agar (TSBA/5; Difco Laboratories, Detroit, MI, USA). For production on solid medium, cells were then restreaked onto TBSA/5, incubated at 28°C for 18 h, removed from the agar surface using sterile cotton swabs, and suspended in weak P0 4 buffer. For production in liquid medium, either 115 strength Tryptic soy broth (TSB/5; Difco Laboratories, Detroit, MI, USA) or a semi-defined complete liquid medium (SDCL) were utilized (Slininger et al., 1994). Washed 24 h old cells from 50 ml precultures (125 ml flask) were used to inoculate 200 ml of experimental culture (500 ml flask). Cultures were incubated at 25°C and shaken at 250 rpm (2.5 cm eccentricity) in a shaker incubator for 96 h. Cells from experimental cultures were then pelleted by centrifugation and washed once in weak P0 4 buffer. Just prior to use, slightly turbid suspensions of antagonist cells from liquid or solid media were adjusted to a common optical absorbance of 0.170 at 620 nm wavelength light (A620). Prior to tuber inoculation, conidial and bacterial suspensions were mixed (l : 1) with final concentrations of 5 x 10 5 conidia/ml and I x 10 8 cfu/ml, respectively, unless otherwise noted. Tubers were inoculated, incubated, harvested, and evaluated for disease symptoms according to methods described by Schisler and Slininger (1994). Briefly, tubers were wounded uniformly at four sites spaced at equal distances around the tubers' circumferences using a blunted steel nail. Wounds were inoculated with 5 pi of a mixed suspension of conidia of an isolate of G. plllicaris and cells of a bacterial antagonist. Controls consisted of wounds that were inoculated with weak P0 4 buffer or suspensions of macroconidia (5 x 10 5 conidia/ml). Tubers were assessed after incubation for 3 weeks in covered trays at 15°C. Disease severity was evaluated by quartering the tubers longitudinally through each of the four wounds and measuring from a wound site, the total depth and width of exposed, necrotic tissue. Experiments on the influence of the production medium on antagonist efficacy were run as 2 x 5 x 5 factorial designs, where levels were two media, five antagonists, and five G. plllicaris isolates. Five additional treatments of each isolate of G. plllicaris inoculated alone acted as controls. This design was repeated in experiments using a second group of five isolates of G. plllicaris (Table 2). Experiments on the influence of potato cultivar on antagonist efficacy were run as 3 x 5 x 5 factorial designs, where levels were three potato cuitivars, five antagonists, and five G. plllicaris isolates. An additional 15 treatments (controls) consisted of each isolate of G. plllicaris tested alone on each of the three potato cultivars. In all trials, there were four replicate wounds/treatment except for 16 replicate wounds for 'G. plllicaris only' controls. Four replicate wounds/treatment occurred once on each of four different potato tubers. Data were subjected to analysis of variance (ANOVA) and means separated using Fisher's protected LSD test (P'::; 0.05). Values from experiments were logtransformed prior to statistical analysis and back-transformed means are reported. All experiments were conducted twice and data from repeated, identical experiments were pooled since treatment by experiment 'interactions were rarely significant. In order to rank antagonist strains' performance relative to each other when tested against pathogen isolates that differed in virulence, relative performance indices of strain efficacy (RPIefficacJ were calculated for each strain's performance against each pathogen isolate when testIng the effect of production medium or host cuitivar on dry rot development. The 10 RPIefficacv values obtained were then used to calculate average RPlefficacv values for each antagonist strain as well as for ANOVA and mean separation of antagonist RPIefficacy values. RPIs allow different types of data to be compared using a standard scale and are dimensionless values that will theoretically range from '0' to '100'. Higher RPIefficacv values indicate superior antagonist strain efficacy compared to strains with lower RPIefficac; values. Assuming dry rot disease severity measurements that are normally distributed across a factor group tested, 'F' wiII range from 2 to + 2 where F= (X - Xavg)/s. Here, X is a


271

single disease severity measurement observed for a strain, and Xavg and s are the average and standard deviation, respectively, of all measurements obtained for all strains in a group. RPIefficac) then is calculated by: I(F - 2) 1*25. Influence of Production Media on Antagonist Efficacy and Rank To determine if the production medium used to produce antagonist biomass would influence the efficacy or RPIefficaev ranking of antagonists, two media, differing both in nutrient composition and phase were utilized to produce biomass of the five bacterial antagonists of dry rot (Table I). Antagonist biomass was produced on either solid Tryptic soy broth (TSBA/5, described above) or a semi-defined complete liquid medium (SDCL, described above). Bioassays of antagonist efficacy against 10 isolates of G. plilicaris (Table 2) on potato tubers (cuitivar Ranger Russet) were then conducted using a 2 x 5 x 5 experimental design (described above), and disease severity and RPIefficac) values were calculated. Influence of G. pulicaris Isolate on the Efficacy Ranking of Dry Rot Antagonists Efficacy data for antagonists grown in SDCL in the above experiment was considered separately. To ascertain whether the ranking of antagonist efficacy varied depending on the isolate of G. plilicaris used in a bioassay, RPIefficacv values obtained from antagonist bioassays against each of the 10 isolates of G. plilicaris were used to determine 10 separate rankings and a cumulative ranking of the antagonist strains. Influence of the Phase of Nutritionally Similar Production Media on Antagonist Efficacy and Rank Experiments were conducted to determine if the phase (solid vs liquid) of the medium used to produce biomass of antagonists influenced antagonist efficacy and ranking when the nutrient composition of the solid and liquid medium was identical. Bioassays of antagonist efficacy against 10 isolates of G. plilicaris (Table 2) on potato tubers (cultivar Russet Burbank) were conducted using a 2 x 5 x 5 experimental design (described above). Disease severity and RPIefficac) values for ranking antagonist strains were calculated. Influence of Potato Cultivar on the Efficacy Ranking of Dry Rot Antagonists Antagonists were tested on potato tubers of three different varieties to determine if the potato cuitivar used in bioassays influenced antagonist efficacy and ranking. Antagonist biomass was produced on SDCL medium, and antagonist efficacy against 10 isolates of G. plilicaris (Table 2) was determined using potato tubers of varieties Russet Burbank, Red Norland, and Yukon Gold. Experiments were conducted using a 3 x 5 x 5 factorial design and disease severity and RPIefficacy values were calculated. RESULTS Influence of Production Media on Antagonist Efficacy and Rank For experiments conducted against the first five isolates of G. plilicaris (group A, Table 2), the medium used to produce the antagonists did not influence antagonist performance as antagonists from either medium reduced disease severity (P:( 0.05, Table 3). Antagonist production medium did influence antagonist efficacy in bioassays against the second five isolates of G. pulicaris (group B, Table 2) as antagonists produced in the liquid medium SDCL effectively reduced disease severity, but to a lesser extent than antagonists produced on the solid medium TSBA/5 (Table 3). For some antagonists, the production medium made a great difference in the RPIefficacv-based ranking of the strain (Table 4). For example, Enterobacter sp. S11 :P:08 was ranked as the most effective of the five strains when antagonists were produced on the solid medium, but ranked as the fourth out of five strains when antagonists were produced in the liquid medium (Table 4).

272


TABLE 3.

Effect of culture medium on antagonist efficacy when antagonists were produced in a semi-defined complete liquid medium (SDCL). or on 1/5 strength tryptic soy broth agar (TSBAI5), and bioassayed against 10 isolates of G. plilicaris" on potato tubers (cv. Ranger Russet) Colonized tuber tissue (mm)"

Treatment G. plilicaris control SDCL antagonists TSBAI5 antagonists

G. plilicaris Group A

G. plilicaris Group B

24.3 a'

25.9a 15.6 b 10.9c

Il.Ob 10.7b

a The 10 isolates of G. plilicaris were split into two groups of five (Groups 'A: and 'B', Table 2) and assayed in separate experiments. "Tubers were quartered 21 days after inoculation and incubation at 15°C by making longitudinal cuts perpendicular to the tuber surface and through previously inoculated wounds. The sum of the depth and width of the exposed darkened, dry rotted tissue was determined. 'Within a column, values followed by unlike letters are significantly different (Fisher's protected LSD, P';; 0.05). ANOYA and mean comparisons were performed on log-transformed data. Backtransformed values are presented.

TABLE 4.

Efficacy relative performance indices (RPl dfic ",) when bacterial antagonists were grown in semi:defined complete liquid medium (SDCL), or on 1/5 tryptic soy broth agar (TSBA/5) and bioassayed against 10 isolates of G. plilicaris on potato tubers (cv. Ranger Russet)

Medium Antagonist S09:Y:08 SII:P:08 SII:P:12 SII:P:14 SII:T:07

SDCL

TSBAI5

56.9 111 a" 46.7 (4 )b 46.4 (5 )b 48.9 (3 ) b 51.1 (2) ab

49.3 (4) ab 55.011 I a 42.5 151 b 50.6(3 ) a 52.6(2 ) a

a Rankings of the RPI"rkoc,. values of the five antagonists were assigned based on results of bioassays using each of 10 isolates of G. plilicaris. "Within a column. values followed by unlike letters are significantly different (Fisher's protected LSD. P ,;; 0.05).

Influence of G. pulicaris Isolate on the Efficacy Ranking of Dry Rot Antagonists

When antagonists were grown in SDCL medium. all five antagonists effectively reduced disease severity when tested against group A and group B isolates of G. plIlicaris (P < 0.05, Table 5), but rarely differed from each other when disease severities were averaged across the five isolates of G. plIlicaris. Antagonist rankings based on RPleffieac\ varied greatly depending on the isolate of G. plIlicaris used in the bioassay. When antagonists were assayed against G. plllicaris 15-3, antagonist P. jlllorescens SII :P: 12 ranked as the most efficacious of all the antagonists yet was the least effective of all strains by a wide margin when bioassayed against G. plIlicaris strain 34-4 (Table 6). When produced in the liquid medium SDCL, antagonist P. jlllorescens S09:Y:08 had the highest rank when RPlefficacy results \vere pooled across all 10 isolates of G. plIlicaris (Table 6).


TABLE 5.

273

Efficacy of bacterial antagonists when grown in semidefined complete liquid medium and assayed against 10 isolates of G. puticaris" on potato tubers (cv. Ranger Russet) Colonized Tuber Tissue (mm)

Treatment G. pulicaris control S09:Y:08 SII :P:08 SI1:P:12 Sll:P:14 Sll:T:07

G. puticaris Group A

G. puticaris Group B

24.3 7.1 c 12.8 b 13.4b 12.3 b 10.6 b

25.9a 14.4 b 16.8b 16.9b 15.0b 14.9b

a The 10 isolates of G. puticaris were split into two groups of five (Groups 'A and 'B', Table 2) and assayed in separate experiments. b Within a column, values followed by unlike letters are significantly different (Fisher's protected LSD, P:( 0.05). ANOVA and mean comparisons were performed on log-transformed data. Backtransformed values are presented.

Influence of the Phase of Nutritionally Similar Production Media on Antagonist Efficacy and Rank Antagonists reduced disease severity caused by G. pulicaris isolates in group A and group B regardless of the production medium utilized (Table 7). Antagonists produced on the solid medium TSBA/5 consistently reduced disease severity to a greater extent than when the same antagonists were produced on the nutritionally identical liquid medium TSB/5 (Table 7). The phase of the nutritionally similar production media made little difference in the RPIefficacv-based ranking of the strains (Table 8), with the first, fourth, and fifth ranked strains being identical for the two production media. Influence of Potato Cultivar on the Efficacy Ranking of Dry Rot Antagonists When bioassayed on three different potato cultivars, antagonists again substantially reduced disease severity caused by either isolate group A or B of G. pulicaris (Table 9). Cultivar had a moderate effect on the RPIcfficacv rankings of antagonists (Table 10). For example, P. fiuorescens SII :P: 14 ranked first, second, or third depending on the cultivar used in the bioassay. RPIcfficacv rankings were identical for antagonists E. cloacae SII :T:07 (fifth) and Enterobacter sp. si I :P:08 (fourth) for bioassays conducted on each of the three cultivars. P. fiuorescens S09:Y:08 achieved the highest RPIcfficacv ranking when results were pooled across all three cultivars (Table 10). .

DISCUSSION For conducting bioassays of biocontrol antagonists, the choice of antagonist production medium, isolate of pathogen, and potato cultivar were all shown to influence the efficacy ranking of antagonist strains. Even among the five antagonist strains that were top ranked based on liquid culture growth kinetics and efficacy against G. pulicaris (Slininger et al., 1994), we were able to demonstrate considerable variation in strain rankings depending on the antagonist production medium, pathogen isolate, and host cultivar used in the bioassays. Accordingly, investigators that initiate research to develop a biocontrol product should carefully consider choices regarding antagonist production medium, pathogen isolates and host cultures when designing antagonist selection experiments. For example, using a medium that is commercially untenable to produce candidate strains of antagonists for bioassays could result in a strain obtaining a comparatively higher efficacy ranking than it \vould

Iv -.J

.f:.

TABLE 6.

Eflicacy relativc pcrformance indices (RPldfic"c,) lor bacterial antagonists grown in semi-defined complete liquid medium and assayed against 10 isolates of G. puticuris on potato tubers (cv. Ranger Russet) RPI~~!:>i~::t:I;

G. pulicaris isolate 10 Isolate Antagonist S09:Y:OS SII:P:OS SII:P:12 SII:P:14 SII:T:07

15-3

34-4

47.41'fl 55.6 (1 ) 50.7 (2 ) 4S.9 1.1)

69.2 111 54.5 1.11 33.2 (5 ) 37.4 14 ) 55.6 (2 )

41-1

AICaND5

KaAFII

LaMc2

NoBe5

NoT&S4

SJlS4C

SJ561 B

avcragc b

47.SI.1I 40.7 (5 ) 41.5 141 54.4 (2 )

60.1 111 44.6141 40.4 (5 ) 55.4 (2 ) 49.6(3)

63.7 111 36.7 (5 ) 55.5(2) 4S.3 1.1) 45.S (4 )

57.6 (1 ) 45.S 141 55.7 12 ) 50.S I .11 40.1 (5 )

41.4 H ) 40.S 151 51.7(2 ) 50.91.1) 65.2 111

73.2111 43.1 141 39.5 151 49.0 (2 ) 45.4 1.1)

44.5 HI 56.S 111 41.4 (5 ) 56.5 (2 ) 5 I.()I.1)

46.2 (5 ) 49.9 1.11 50.S (2 ) 4S.6 141 54.7 (1 )

56.9 (1)a 46.7141 b 46.4151 b 4S.91.1)b 51.1 12 )ab

"Rankings of the RPl dfim , values of the five antagonists were assigned based on results of bioassays using each of 10 isolates of G. pulicaris. The 'I O-isolate average' rank was assigned aner averaging the 10 RPld1lc"cy values. /> Within the column, values lollowed by unlike letters are significantly dilTerent (Fisher's protected LSD, P =( 0.(5).

p ?' Vl n

:c

Vi t""

I"!'I

;xi

t"l

'-oj

,.."'


TABLE 7.

275

Effect of liquid (TSB/5) and solid (TSBA/5) medium (l/5 strength tryptic soy broth or agar) on the efficacy of dry rot antagonists assayed against 10 isolates of

G. plilicaris· on potato tubers (cv. Russet Burbank) Colonized tuber tissue (mm) Treatment G. plilicaris control TSB/5 antagonists TSBA/5 antagonists



29.7 3.2b l.5c

46.2a 7.0b 4.7c

• The 10 isolates of G. plilicaris were split into two groups of five (Groups 'A' and 'B', Table 2) and assayed in separate experiments. b Within a column, values followed by unlike letters are significantly different (Fisher's protected LSD. P ~ 0.05). ANOVA and mean comparisons were performed on log-transformed data. Backtransformed values are presented.

TABLE 8.

Efficacy relative performance indices (RPI"rk"') when bacterial antagonists were grown in liquid (TSB/5) or solid (TSBA/5) forms of liS strength tryptic soy medium and bioassayed against 10 isolates of G. plIlicaris on potato tubers (cv. Russet Burbank)

Medium Antagonist S09:Y:08 SII:P:08 SII:P:12 SII:P:14 SII:T:07

TSB/5

TSBA/5

58.6(1) a 45.8 151 b 50.412 ) ab 48.8 (3 ) b 46.4 (4 ) b b

59.1(1l a 44.8 (5 )b 49.7 (3 ) b 51.1 (2 ) ab 45.414 ) b

a Rankings of the efficacy RPI"r;m, values of the 5 antagonists were assigned based on results of bioassays using each of 10 isolates of G. pulicaris. b Within a column, values followed by unlike letters are significantly different (Fisher's protected LSD, P ~ 0.05).

when produced in a commercially viable medium. Our results indicate that an accurate ranking of antagonists also requires that they are bioassayed against a representative sample of pathogen isolates and host cultivars. It is obvious that diminishing returns regarding increased accuracy of ranking candidate biocontrol strains could be expected if limits are not placed on the number of media types, pathogen isolates, and host cultivars evaluated. By carefully evaluating types of media in light of commercial requirements, biocontrol agents against isolates of the target pathogen that are representative of those encountered in prospective areas of biocontrol agent deployment, and biocontrol efficacy of strains on host cultivars that make up a preponderance of those commercially produced, one could theoretically approach maximal accuracy in ranking prospective biocontrol strains without conducting an unreasonable number of bioassays. When identical nutrient media that differed only in phase were used to produce antagonists, antagonists were more effective when produced on the solid medium (Table 7), but the

276


TABLE 9.

Efficacy of bacterial antagonists when assayed against 10 isolates of G. plilicaris" on potato tubers of three cultivars"

Treatment G. plilicaris only S09:Y:08 SII:P:08 SII:P:12 SII:P:14 SII:T:07



27.4a e 5.1 d 9.0b 5.7cd 6.1 cd 7.6bc

16.9a 4.8c 8.0b 5.0c 5.1 c 7.0b

a The 10 isolates of G. plilicaris were split into two groups of five (groups 'N and 'B', Table 2) and assayed in separate experiments. "Table values represent data pooled from assays using potato cultivars Russet Burbank. Red Norland. and Yukon Gold. e Within a column. values followed by unlike letters are significantly different (Fisher's protected LSD, P ~ 0.05). ANOVA and mean comparisons were performed on log-transformed data. Backtransformed values are presented.

TABLE 10.

Efficacy relative performance indices (RPI,rfimy) when bacterial antagonists were assayed against 10 isolates of G. plilicaris on potato tubers of three cultivars RPI~rrfrc~)~y

Antagonist S09:Y:08 SII:P:08 SII:P:12 SII:P:14 SII:T:07

Russet Burbank 51.1 I}I abe 43.6 151 c 53.6121 ab 54.3 (1 ) a 47.5 141 bc

Red Norland

Yukon Gold

Three cultivar average

53.8 (1) a 45.6 151 a 53.6121 a 50.5 1}1 a 46.6 141 a

61.8 (1) a 39.6 151 C 51.0131 b 52.1 121 b 45.6 141 bc

55.6 111 a 42.9 151 b 52.7(2) a 52.3(3) a 46.5 141 b

"RPl cffim , values were calculated for each cultivar (3) by G. plilicaris strain (10). Thus each table value represents the mean of 10 RPI,rr"", values. "Within a column, values follO\ved by unlike letters are significantly different (Fisher's protected LSD,

P ~0.05).

RPlcfficacv ranking of strains was similar regardless of the phase of the medium (Table 8). While thIS demonstrates that the phase of the production medium will not necessarily affect the efficacy ranking of antagonists, the importance of the phase of a production medium in influencing efficacy ranking cannot be dismissed on the basis of the present results. The five antagonists tested in this study were the best of 18 strains in efficacy and growth kinetics when produced in liquid culture (Slininger et al., 1994). However, some of the 18 strains of bacteria did not grow well in liquid culture, such as P. corrllgata S09:P:06 (Schisler & Slininger, 1997). The lack of effectiveness and poor growth kinetics of this strain when produced on two different liquid media indicates that its RPlcfficacy ranking would have been adversely affected had it been produced in liquid rather than solid media, and included in this study's group of antagonists. Liquid culture growth kinetics vary between bacterial strains and is another critical factor that should be evaluated when devising a method for identifying biological control strains with a maximum potential for commercial development (Slininger et al., 1994; Schisler & Slininger, 1997). The isolate of G. plilicaris used in a bioassay often determined whether the efficacy ranking of an antagonist was first, fifth, or in between. Only after results from all 10 isolates of G. plilicaris were averaged did trends in rankings become apparent (Table 6). These results


277

demonstrate the risk in selecting supposedly superior biological control strains based on bioassays conducted against a single isolate of a pathogen. In the present study, the most efficacious strain would have erroneously appeared to be Enterobacter sp. Sll:P:08 had the bioassay only been conducted against G. pulicaris SJl84C (Table 6). As pathogen surveys may not exist, or be limited in scope for a particular pathosystem being studied, an accurate ranking and selection of the best antagonist for development may require the researcher to select pathogen isolates from those areas in which a prospective biocontrol product would be utilized. Our current results indicate that randomly choosing a biocontrol strain from one location, or a culture collection, could easily result in choosing a biocontrol strain for development that is not the best choice for controlling the variety of pathogen isolates present in the prospective area of deploying a biocontrol product. For those tested, the host cultivar used for bioassays of antagonist efficacy had a modest influence on the ranking of antagonists (Table 10). Interestingly, Enterobacter sp. SII:P:08 ranked first of five antagonists when grown on TSBA/5 and assayed on Ranger Russet tubers (Table 4), but in separate experiments ranked last when grown on the same medium and assayed on tubers of Russet Burbank (Table 10). Thus, it appears that the degree of importance a host cultivar makes in the efficacy ranking of antagonists will be dependent on what host cultivars are used in bioassays. In this study, we have not attempted to determine the mechanisms by which the factors of cultivation medium, pathogen strain, and host cultivar modify the efficacy of antagonists. Several interesting possibilities for further study exist. The nutritional composition of a medium used to produce biomass of an antagonist is known to influence secondary metabolite production and the production of known antibiotic compounds that can playa key role in biological control (Dickie & Bell, 1995; Slininger et al., 1997). This is likely to be a factor that contributes to the differences in antagonist efficacy rankings seen in this study. Complicating how the role of production medium might impact biocontrol strain efficacy and ranking is the fact that different isolates of G. pulicaris are known to produce differing levels of fusaric acid (Bacon et af., 1996), a compound that limits the production of the antibiotic 2,4-diacetylphloroglucinol by some strains of antagonists but not others (Duffy & Defago, 1997). Similarly, the phytotoxin enniatin is also produced in differing amounts by different isolates of G. pulicaris (Herrmann et af., 1996). This could modify biocontrol agent activity depending on a strain's sensitivity to the compound since enniatin is known to have antibiotic properties (Grove & Pople, 1980). Whether the production of differing levels of these compounds by the isolates of G. pulicaris used in this study accounts for a portion of the differences in antagonist efficacy ranking when bioassayed against different strains of G. pulicaris is unknown. How the host cultivar influences the efficacy ranking of dry rot antagonists is also unknown. In another pathosystem, Nelson et al. (1986) found that variability in the biological control of Pythium ultimum by several strains of E. cloacae was associated with differences in the nutrient profiles of the rhizosphere exudates of different species of host plants. The strains of E. cloacae exhibited better efficacy in reducing disease on plant species that had low levels of carbohydrates in the rhizosphere. As the nutrient content of potato cultivars can vary (Talley et al., 1983), it is conceivable that differences in nutrient content between cultivars also contributes to differences in biocontrol efficacy rankings when antagonists are bioassayed on different cultivars. We have demonstrated that the medium used to produce biological control agents, the isolate of a pathogen used in a bioassay and the cultivar of the host all influence both the efficacy of an individual biological control strain and, importantly, the performance of the strain relative to other strains being tested. It is well documented that results of ill vitro antagonist screens of putative biocontrol agents can be widely divergent depending on the conditions utilized in the bioassay, including growth medium, strain of pathogen, and temperature (Dickie & Bell, 1995). Our current work demonstrates that even the results of ill situ bioassays of biocontrol agent performance will be dependent on the choice of antagonist cultivation medium, pathogen isolate, and host cultivar. By conducting biocontrol

278


bioassays that use a range of each of these factors and ensuring that the choices of antagonist production media, pathogen isolates, and host cultivars accurately reflect the conditions under which prospective biocontrol agents will be commercially produced and deployed, one can greatly increase the accuracy of choosing a strain that has maximum potential for successful commercial development.

ACKNOWLEDGEMENTS Excellent technical assistance was provided by Jennifer lemHi and Patricia Soncasie.

REFERENCES BAKER, K.F. (1987) Evolving concepts of biological control of plant pathogens. Annual Review ofPhytopathology 25, 67-85. BACON, CW.• PORTER, J.K., NORRED, W.P. & LESLIE, J.F. (1996) Production of fusaric acid by Fusarium species. Applied and Environmental Microbiology 62, 4039--4043. COOK. R.J. (1993) Making greater use of introduced microorganisms for biological control of plant pathogens. Annual Review of Phytopathology 31, 53-80. DICKIE, G.A. & BELL CR. (1995) A full factorial analysis of nine factors influencing in vitro antagonistic screens for potential biocontrol agents. Canadian Journal of lvficrobiology 41, 284-293. DUFFY, B.K. & DEFAGO, G. (1997) A Fusarium pathogenicity factor blocks antibiotic biosynthesis by antagonistic pseudomonads. Phytopathology 87, S26. DUFFY. B.K. & DEFAGO. G. (1999) Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas j/uorescens biocontrol strains. Applied and Environmental i\licrobiology 65, 2429-2438. FRAVEL. D.R. & LARKIN, R.P. (1996) Availability and application of biocontrol products. Biological and CullUral Tests 11, 1-7. GROVE, J.F. & POPLE, M. (1980) The insecticidal activity of beauvericin and the enniatin complex. lvfycopathologia 70, 103-105. HANSON, L.E .• SCHWAGER, SJ. & LORIA, R. (1996) Sensitivity to thiabendazole in Fusarium species associated with dry rot of potato. Phytopathology 86, 378-384. HERRMANN, M., ZOCHER, R. & H.-\.ESE. A. (1996) Enniatin production by Fusarium strains and its effect on potato tuber tissue. Applied and Environmental J\;ficrobiology 62, 393-398. KING, E.B. & PARKE, J.L. (1993) Biocontrol of Aphanomyces root rot and Pythium damping-off by Pseudomonas cepacia AMMD on four pea cultivars. Plant Disease 77, 1185-1188. KING, E.B. & PARKE, J.L. (1996) Population density of the biocontrol agent Burkholderia cepacia AMMDRI on four pea cultivars. Soil Biology & Biochemistry 28,307-312. KINKEL, L.L. (1997) Microbial population dynamics on leaves. Annual Review of Phytopathology 35,327-347. KNUDESN, LM.B., HOCKENHULL. J .• FUNCK JENSEN, D .• GERHARDSON, B., HOKEBERG. M .• TAHVONEN, R., TEPERI. E., SUNDHEIM, L. & HENRIKSEN, B. (1997) Selection of biological control agents for controlling soil and seed-borne diseases in the field. European Journal of Plant Pathology 103, 775-784. NELSON, E.B., CHAO, W.-L., NORTON, J.M., NASH, G.T. & HARMAN, G.E. (1986) Attachment of Enterobacter cloacae to hyphae of Pythium ultimum: possible role in the biological control of Pythium preemergence damping-off. Phytopathology 76, 327-335. PIERSON, L.S. III. WOOD, D.W. & PIERSON, E.A. (1998) Homoserine lactone-mediated gene regulation in plant-associated bacteria. Annual Review of Phytopathology 36, 207-225. SCHISLER. D.A. & SLININGER, PJ. (1994) Selection and performance of bacterial strains for biologically controlling Fusarium dry rot of potatoes incited by Gibberella pulicaris. Plant Disease 78, 251-255. SCHISLER, D.A. & SLININGER. P.J. (1997) Microbial selection strategies that enhance the likelihood of developing commercial biological control products. Journal of Industrial and J'vficrobiology and Bioteclmology 19,172-179. SCHISLER, D.A., SLININGER, P.J. & BOTHAST, R.J. (1997) Effects of antagonist cell concentration and twostrain mixtures on biological control of Fusarium dry rot of potatoes. Phytopathology 87, 177-183. SCHISLER, D.A., SLININGER, P.J., KLEINKOPF, G .• BOTHAST, R.J. & OSTROWSKI, R.C (2000) Biological control of Fusarium dry rot of potato tubers under commercial storage conditions. American Joumal of Potato Research 77, 29--40. SLININGER. P.J., VAN CAUWENBERGE. J.E., SHEA-WILBUR, M.A., BURKHEAD. K.D., SCHISLER. D.A. & BOTHAST. R.J. (1997) Reduction of phenazine-l-carboxylic acid accumulation in growth cultures of the biocontrol agent Pseudomonas j/uorescens 2-79 eliminates phytotoxic effects of wheat seed inocula without sacrifice to take-all suppressiveness. Plant Growth Promoting Rhizobacteria. Present Status and Future Prospects, 4th International Workshop on Plant-Growth promoting Rhizobacteria in Sapporo. Japan


279

(OGOSHI el al., Eds), 4th PGPR International Workshop Organizing Committee: Faculty of Agriculture, Hokkaido University, Sapporo, pp. 464-467. SW,;jNGER, P.l., SCHISLER, D.A. & BOHIAST, RJ. (1994) Two-dimensional liquid culture focusing: a method of selecting commercially promising microbial isolates with demonstrated biological control capability. Improl'ing Plml! Produclivily willI RhizosplIere Bacleria, 3rd International Workshop on Plant GrowthPromoting Rhizobacteria, Adelaide, S. Australia (RYDER, M.H. el al., Eds), CSIRO Division of Soils, Glen Osmond, pp. 29-32. TALLEY, E.A., TOMA, R.B. & ORR, P.H. (1983) Composition of raw and cooked potato peel and flesh: amino acid content. Journal of Food Science 48, 1360-1363.