A Plant Growth Promoting Rhizobacterium and Temperature Effects on Performance of 18 Clones of...

Amer J of Potato Res (1998) 75:145-152 145

A Plant Growth Promoting Rhizobacterium and Temperature

Effects on Performance of 18 Clones of Potato

Salah Bensalim, J e r zy N o w a k 1, and Samuel I~ Asiedu

1Affiliation: Department of Plant Science, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3. Corresponding author. Correspondence/communication address: Dr. Jerzy Nowak, Department of Plant Science, Nova Scotia A~ricultural College,

Truro, Nova Scotia~ Canada B2N 5E3; Tel: (902) 893-6686; Fax: (902) 897-9762; E-mail: [email protected]

ABSTRACT

A survey of genotypic responses to beneficial bac-

ter ium ( P s e u d o m o n a s sp. s train PsJN) was conducted

in v i t ro and e x v i t ro , unde r two t empe ra tu r e condi-

tions, using eighteen clones of pota to o f different heat

s t r e ss to le rance : t e m p e r a t e adap ted cul t ivars Ken-

nebec and Russet Burbank; heat to lerant DTO-2, DTO-

28, DTO-33, LT-1, LT-2, LT-5, LT-6, LT-7, LT-8, LT-9,

Y84-02, NDD277-2, D~sir~e, and Maine-47; and hea t

s ens i t i ve absc i ss ic ac id (ABA)-de f i c i en t m u t a n t s

11401-01 and 9120-05. Nodal explants t aken f rom 6-

week -o ld b a c t e r i z e d and n o n - b a c t e r i z e d c o n t r o l

p l an t l e t s were cu l tu red in v i t ro on a ho rm one - f r ee

p o t a t o nodal cu t t ing medium, and p laced a t e i t he r

20/15 C or 33/25 C day/night t empera tu re , 12h photoper iod and 250 pE m -2 s-1 mixture of f luorescent and

incandescen t light, fo r six weeks. The t ube r i za t i on

response was studied ex vi tro af ter two weeks accli-

mat ion of 2-week old p lant le ts a t 33/25C. The accli-

m a t e d p l a n t l e t s w e r e t r a n s p l a n t e d t o 3L p las t i c

nurse ry pots containing peat-based Pro-Mix growing

medium and placed in growth chambers a t e i ther 20/15

or 33/25 C day/night t empera ture , 12 h photoper iod,

475 IrE m 2 s ~ light and =80% RH, for 12 weeks. Com-

pared to the non-bacterized controls, bacterizat ion sig-

nificantly increased stem length of 12, shoot biomass of

9, and roo t biomass o f 2 clones at 20/15C; and s tem

length of 14, shoot biomass o f 15, and root biomass of

13 clones at 33/25C. High temperature increased length

o f internodes and had e i ther no effect or slightly de-

creased node number. Tempera ture increase had the

most dramatic effect on roo t development. An average

shoot to root rat io decreased from 3.7 at 20/15 C to 1.7

Accepted for publication February 9, 1998. ADDITIONAL KEY WORDS: Beneficial bacterium, heat tolerance, Solanum ~berosum, in vitro, ex vitro.

at 33/25 C for non-bacter ized p lant le ts and, respec-

tively, f rom 4.3 to 1.5 for bacter ized. The beneficial

effect o f bacter izat ion on roo t biomass was the most

pronounced in LT-1 and Maine-47 a t 20/15 C and LT-8,

Maine 47, DTO-2, Kennebec, NDD277-2 and 11401-01

a t 33/25C. The t empera tu re e levat ion did no t signi-

f i c an t ly a f f e c t r o o t b iomass o f LT-6, DTO-28 and

Ddsir~e. Temperature stress caused severe reduct ion

in tuber number and tuber fresh weight. ABA-deficient

mutan t s did no t produce any tubers and LT-8, LT-9,

Y84-027 and DTO-28 tuberized very poorly at 33/25C.

DTO-33, D~sir~e, LT-1 and Kennebec gave the highest

number o f tubers per pot and Kennebec, LT-1, D~sir~e

and LT-7 the highest yields a t this temperature. There

was no s ignif icant effect o f bac te r i za t ion on tuber-

izat ion a t 20/15 C but at 33/25 C bacterizat ion signifi-

cantly enhanced tuber number and weight in LT-7 and

reduced tuber weight in DTO-2. Although there was no

clear link between the in v i tro response of part icular

clones to bacterization and the i r heat stress tolerance,

improvement of ex vi tro performance of heat to le rant

LT-7 indicates tha t rh izosphere bac te r ia may play a

role in clonal adaptat ion of po ta to to heat stress.

INTRODUCTION

Heat stress tolerance in potato is strongly influenced by

genotype and environment (Mendoza and Estrada 1979, Mid-

more 1992). Availability of water (Trebajo and Midmore

1990), humidity (Mendoza and Estrada 1979), irradiance

(Menzel 1985), photoperiod and nitrogen are considered the

most critical environmental factors affecting response to

heat in potato (reviewed in Ewing 1995). in vitro experi-

ments also indicate differential responses of potato clones

to constituents of growing media (Nowak and Colborne

1989, Nowak and Asiedu 1992), and beneficial bacteria

(Nowak et al. 1995 and 1997, Conn eta/. 1997). To our knowl-

edge, there is no published information on the role of rhizo-

sphere microflora in heat responses of potato.

146 AMERICAN JOURNAL OF POTATO RESEARCH Vol. 75

Beneficial bacteria, designated as plant-growth promoting rhizobacteria (PGPR) (Kloepper et al. 1988), colonize

roots, enhance shoot emergence, and stimulate plant growth either directly, by producing plant hormones and improving

nutrient uptake, or indirectly, by changing the microbial bal-

ance in the rhizosphere in favor of the beneficial microorganisms (reviewed by: Brown 1974, Kloepper et al. 1988,

Glick 1995, Lazarovits and Nowak 1997). A non-fluorescent PGPR, P s ~ sp. strain PsJN, isolated in our labora-

tory, increases root branching, root and shoot dry weights, node number, stem length and lignification, leaf pubescence

and chlorophyll content in tissue culture grown potato plantlets (Frommel et al. 1991). Bacterized plantlets have

fully functioning stomata (Frommel et al. 1991) and tolerate transplant stress much better than non- bacterized controls

(Nowak et al. 1995, Nowak et al. 1997). Furthermore, they have higher cytokinin content and, after transplanting, initi-

ate stolons and tubers earlier than non-bacterized controls

(Lazarovits and Nowak 1997, Dunbar 1997). Clonal variation in the bacterium stimulation of root sys-

tem development, both, i n vitro (Conn et al., 1997) and ex vitro (Dunbar, 1997), and overall improvement of transplants

water management (Nowak et al., 1995; Lazarovits and Nowak, 1997) in particMar, lead us to hypothesize that rhizo-

sphere bacteria modify potato responses to heat stress. The objective of this study was to survey bacterization effects on

i n vitro and ex vitro performance of potato clones of varied heat stress tolerance under elevated temperatures.

MATERIALS AND METHODS

P l a n t Mater ia l a n d Cul ture

Disease-indexed tissue culture plantlets of 14 potato

clones selected for heat tolerance, LT-1, LT-2, LT-5, LT-6, LT- 7, LT-8, LT-9, DTO-2, DTO-28, DTO-33, NDD277-2, Y84-027, D6sir6e, and Maine-47, were obtained from the International

Potato Centre (CIP), Lima, Peru. Plantlets of two temperate adapted cultivars, Kennebec and Russet Burbank, were from

the Plant Propagation Centre, New Brunswick Department of Agriculture, Fredericton, NB, Canada. Two abscissic acid (ABA)-deficient mutants, diploids, 11401-01 and 9120-05, sim-

ilar to the "droopy" mutant (Simmonds 1965, Quarrie 1982), were supplied as tubers by Dr. Henry de Jong, Agriculture

and Agri-Food Canada Research Station, Fredericton, NB, Canada. Tissue culture of the mutants was initiated in our

laboratory. All clones were cultured in 25 X 150 mm test tubes, using single-node explants, on 10 ml of Murashige and

Skoog based, hormone-free, potato nodal cutting medium

(PNCM) as in Sipos et al. (1988). Unless specified otherwise, plantlets were maintained and multiplied under 16h photo-

period, 160-180 HE m -2 s -1 fluorescent light and 22/19 C day/

night growth room temperature.

I n v i t r o B a c t e r i z a t i o n

A plant growth promoting, non-fluorescent, Pseudomo-

nas sp. strain PsJN, originally isolated from surface sterilized

onion roots by J. Nowak (reported by Herman, 1987), was

maintained i n p lan ta , using i n v i t r o grown Kennebec plantlets. The bacterial inoculum was prepared as in Pillay

and Nowak (1997). About 1 cm long nodal explants, taken from 6-week-old plantlets, were dipped in the inoculum for 30 sec, blotted with sterile filter paper, and placed on PNCM.

Non-bacterized controls were immersed in phosphate buffered saline only. The cultures were grown in the growth

room as above.

Tempera ture a n d B a c t e r i z a t i o n Effects on P lan t l e t Growth

Tempera ture and bacter izat ion effects on i n v i t ro

growth of 18 potato clones were determined using nodal

explants taken from 6-week old bacterized and non-bacterized plantlets. The explants were cultured as above and

placed in two growth chambers, 20/15 C and 33/25 C day/

night temperatures, respectively, and 250 p E m ~ s -1 fluorescent and incandescent lights mixture, at 12h photoperiod. After 6 weeks, plantlets were harvested and stem length,

node number, and shoot (stem and leaves) and root dry weights determined (after forced dry air drying at 60 C for 48h). The experiment was conducted twice.

Ex vitro Tuber i za t ion

Nodal explants of 18 bacterized and non-bacterized clones were grown i n vitro in 20/15 C growth chamber as

above, for two weeks. Two-week-old plantlets of the same size were randomly assigned to two groups and transplanted into 3L plastic nursery pots filled with a peat based Pro-Mix

growing medium (ASB Greenworld, Point Spain, NB,

Canada), one plant per pot. Group I transplants were placed in 20/15 C growth chamber and group II in 33/25C. Light intensity, provided by a mixture of fluorescent and incan-

descent lights and measured with LI-COR quantum sensor, was 475 pE m 2 s-', with 12h photoperiod and =80% RH. All

plants were watered daily, and fertilizer was applied weekly, starting one week after transplanting (WAT). Weekly fertil-

1998 BENSALIM, eta/.: PERFORMANCE OF CLONES OF POTATO 147

ization was as described in Dunbar (1997). During weeks 2-

5, a stock solution of 50 and 20 grams of 10-52-10 and 13-0-44

N:P:K, respectively, and 50 g of magnesium sulphate, and 3

g of the chelated micro-nutrients (Plant Products Co. Ltd.,

Brampton, ON, Canada) was mixed in 1L distilled water

(Stock A). In a separate flask, 20 g of calcium nitrate (15.5-

0-0) was dissolved in 1L distilled water (Stock B). Aliquats of

10 mL of each stock were mixed in 1L water prior to each

fertilization. From week 8 on, 13-0-44 (Stock A) and 15.5-0-0

(Stock B) were both increased to 50 g, and 10-52-10 was

decreased to 20 g (Stock A). To promote tuberization, during

the weeks 6 and 7 the plants did not receive any nutrients.

Fertilization was terminated on week 10. Tubers were har-

vested 12 WAT and tuber number and fresh weight per plant

were determined. The conditions for the experiment above

were established after a preliminary experiment with 11

potato clones and 4 replicates of each combination of treat-

ments was conducted.

E x p e r i m e n t a l D e s i g n a n d D a t a A n a l y s i s

A split plot factorial design, with two temperature levels

(20/15 C and 33/25 C), two bacterization levels (bacterized

and non-bacterized) and 18 potato clones was used to deter-

mine the effects of temperature and bacterization on in vitro

potato plantlet growth. Temperature was assigned to the

main plot, whereas the subplot contained the factorial com-

bination of bacterization and clones. Each treatment combi-

nation was replicated 12 times. To assess tuberization

responses to temperature and bacterization, a factorial

arrangement of two levels of bacterization and two tempera-

tures, with 18 potato clones was used in a randomized com-

plete block design, with 3 replicates. Analysis of variance (ANOVA) was performed using a

computer statistical analysis program (SAS Institute Inc.,

Cary, NC). Treatment means were separated using LSD.05. To

avoid the deflation of the variance in the ex vitro experiment,

ABA-deficient mutants response was excluded from the

ANOVA.

RESULTS

I n v i t r o P l a n t l e t G r o w t h

Temperature X bacterization X clone interactions were

highly significant (P=0.0001) for in vitro growth responses of

FIGURE 1 Ef fec t ofPseudomonas sp. s train PsJN, on growth o f DTO-2 (A),11401-01 (B) , and LT-7 (C) potato clones cultured in vitro at 20/15 C and 33/25C day/night temperatures.


A 1 2 ,

LSD(0 05) = 0 8

11 ~

a. 10

i 9 ,

8 ,

-t I

20 33

B 12 .

LSD(0 05) = 0 8

lO

9 ,

8 ,

7 , I 33

6O

55

50

45

v 40

3o

2O

20

c LSD(0 G5) = 4 9 mg

/

33

4O

35

30

~ zs ~ 2o

I:1

F I G U R E 2

D

Temperature (C ~ 20 33 Temperature (C ~

Temperature X bacterization interaction effect on stem length (A), node number (B) , shoot dry weight (C) , and root dry weight (D) o f 18 clones o f potato co-cultured in vitro with strain PsJN. [ ] Non-bacterized, �9 Bacterized.

the tested potato clones. Response variation is represented by DTO-2, 11401-01, and LT-7 in Fig.1 (& B, and C, respec-

tively). Significant interaction between temperature and bacterization was recorded for stem length (P=0.0352), and shoot (P=0.0013) and root (P=0.0001) dry weights (Fig. 2A~ C and D, respectively). There was no significant interaction between

these two factors and the node number (Fig. 2B). On average, at 20/15C, bacterized plantlets were taller (86%), had more nodes (19%), and contained higher shoot (62~ and root (57~ dry weights than non-bacterized controls (Table 1). The dif-

ference in growth responses between bacterized and control treatments was enhanced at 33/25C; the difference in the biomass accumulation was 162% and 129~ for root and shoot dry

weights, respectively. Compared to 20/15C, at 33f25 C bacterized plants had 3120/5 higher root and 54% higher shoot biomass and 27% longer stems. Node number was, however, 15%

greater at 20/15 C (Fig. 2B). Temperature increase caused reallocafion of biomass from shoot to root portion of plantlets (Table 1); an average value of the shoot to root ratio was 3.7

at 20/15 C and 1.7 at 33/25C. This reallocation was even more dramatic in bacterized plantlets; shoot/root ratio decreased

from 4.3 at 20/15 C to 1.5 at 33/25C. Compared to the non-bacterized controls, bacterization

at 20/15 C significantly increased shoot biomass in 1104-01,

LT-1, LT-5, LT-9, DTO-2, DTO-28, Maine-47, D~sir~e, and

NDD277-2, but not in 9120-05, LT-2, LT-7, LT-8, DTO-33, Ken- nebec, Russet Burbank, and Y84-027 (Table 1). Clones 1104- 01, DTO-2, Y84-027, LT-7 and NDD277-2 benefitted the most

and LT-9, LT-6 and LT-1, the least at 33/25C. The beneficial effect of bacterization on root biomass at

20/15 C was only significant in Maine-47 (160%) and LT-1 (156% stimulation). On the other hand, 13 clones benefitted

significantly from this treatment at 33f25 C (Table 1). Bacter- ized LT-8, Maine-47, DTO-2, Kennebec, NDD277-2 and 11401-

01 had the largest root mass at the elevated temperature and LT-6, DTO-28 and D~sir~e, the smallest. The most pronounced

difference in root biomass between bacterized and control plants at 33/25 C was in DTO-2, Kennebec and 11401-01 (54.9,

42.1 and 39.4 mg per plantlet, respectively). Root growth stimulation at the higher temperature lowered shoot/root (S/R) dry matter ratio in all clones (Table 1); in Y84-027 (278%), Ken-

nebec (250%), LT-8 (221%), LT-9 (207~ and Russet Burbank

(181%) the most and DTO-33 (13~ LT-5 (30~ LT-6 (60%), 11401-01 (66%) and LT-2 (71%), the least. This ratio was lowered even further in clones which best responded to bacterization at 33/25C, i.e. Kennebec (511%), NDD277-2 (391%), LT-8

(308%), Russet Burbank (300~ and 11401-01 (294%). High temperature (33/25C) significantly inc.reased total

biomass (shoot plus root) of non-bacterized plantlets only in LT-8, Maine-47, LT-9, and LT-1. There was no significant dif-

1998 BENSALIM, eta/.: P E R F O R M A N C E O F CLONES O F POTATO 149

TABLE 1.--Effects of temperature and bacterization wi th P s e u d o m o n a s sp. strain PsJN, on in vitro growth responses of 18

potato clones.

Clone SL NN SDW RDW TB S/R Trt T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2

11401-01

9120-05

LT-1

LT-2

LT-5

LT-6

LT-7

LT-8

LT-9

DTO-2

DTO-28

DTO-33

Kennebec

R.Burbank

Maine-47

D~sir~e

NDD277-2

Y84-027

C 3.3 7.0 7.0 10.8 8.0 13.7 2.0 5.8 10.0 19.5 4.0 2.4 B 13.8 12.5 17.2 12.2 63.3 70.2 5.7 45.2 42.0 115.4 6.3 1.6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 5.5 8.1 9.3 10.8 14.5 22.8 2.7 8.3 17.2 31.1 5.4 2.7 B 8.8 15.5 8.4 12.9 25.3 64.3 4.9 28.5 30.2 92.8 5.2 2.3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 5.8 5.7 11.1 6.8 28.3 31.6 9.9 29.3 38.2 60.9 2.9 1.1 B 11.2 8.9 11.3 8.2 52.9 36.6 25.4 34.4 78.3 71.0 2.1 1.1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 5.9 7.6 11.3 10.2 22.3 30.1 7.7 17.7 30.0 47.8 2.9 1.7 B 6.3 9.1 9.9 9.1 24.1 43.5 6.1 34.6 30.2 78.1 3.9 1.3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 6.6 9.8 12.3 9.8 23.0 30.3 6.6 11.3 29.6 41.6 3.5 2.7 B 10.6 13.7 12.8 10.1 36.5 83.8 5.9 48.0 42.4 131.6 6.2 1.8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 4.3 4.9 8.9 7.2 15.4 14.3 6.5 9.3 21.9 23.6 2.4 1.5 B 4.3 5.9 8.5 6.8 16.7 15.7 8.0 9.9 24.7 25.6 2.1 1.6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 2.0 2.2 6.4 4.5 20.7 16.7 4.4 7.3 25.1 24.0 4.7 2.3 B 3.3 9.8 7.8 8.9 30.0 68.4 5.4 37.2 35.4 105.6 5.5 1.8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 8.3 7.4 11.4 8.0 41.3 53.3 9.2 38.6 50.5 91.9 4.5 1.4 B 9.1 13.2 12.1 9.2 48.0 92.7 9.6 75.4 57.6 168.1 4.9 1.2

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 5.8 7.7 11.3 8.1 31.2 37.0 7.8 28.1 39.0 65.1 4.0 1.3 B 11.4 8.3 12.3 8.3 50.1 40.1 11.3 30.7 61.4 70.8 4.4 1.3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 3.8 4.6 11.1 6.3 19.5 15.8 5.0 7.3 24.5 23.1 3.9 2.2 B 9.9 14.2 12.4 11.8 35.2 78.2 9.3 62.2 44.5 140.4 3.8 1.3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 3.1 3.6 7.3 5.9 20.3 14.7 5.2 8.6 25.5 23.3 3.9 1.7 B 8.6 7.0 11.1 7.9 35.3 29.6 8.6 16.6 43.9 46.2 4.1 1.8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 4.1 4.5 9.1 7.1 16.9 22.3 6.8 10.0 23.7 32.3 2.5 2.2 B 6.3 10.9 11.1 10.0 24.3 48.8 6.4 30.1 30.7 78.9 3.8 1.6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 6.2 5.0 12.2 6.6 30.3 23.8 7.2 9.8 37.5 43.6 4.2 1.2 B 10.2 11.7 12.6 9.4 42.0 47.6 7.7 52.3 49.7 99.9 5.5 0.9

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 5.0 4.9 11.3 7.2 25.5 20.2 5.7 12.8 31.2 33.0 4.5 1.6 B 8.4 13.4 12.3 10.1 31.6 54.6 5.7 39.3 37.3 93.9 5.6 1.4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 6.5 8.5 11.9 11.2 38.1 47.4 10.0 28.4 48.1 75.8 3.8 1.7 B 10.0 15.4 11.9 13.5 57.6 77.6 26.0 62.6 83.6 140.2 2.2 1.2

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 3.9 5.4 8.1 6.6 13.4 17.7 4.8 11.7 18.2 29.4 2.8 1.5 B 10.2 10.4 11.0 9.4 40.2 44.8 13.8 21.3 54.0 66.1 2.9 2.1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 2.6 4.8 9.2 8.2 13.9 13.7 3.9 10.4 17.8 24.1 3.6 1.3 B 7.8 8.4 10.0 10.0 30.0 52.1 5.5 47.9 35.5 100.0 5.4 1.1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 3.8 3.5 6.3 6.3 24.0 11.6 7.0 12.0 31.0 23.6 3.4 0.9 B 5.5 9.9 8.7 8.8 32.1 48.0 7.7 35.5 39.8 83.5 4.2 1.4

Values per plantlet are means of 12 replicates. Treatment Crrt): T1, 20/15 C; T2, 33/25 C; C, control; B, bacterized plantlets. LSD 05 values: stem length (SL), 2.9 cm; node number (NN), 3.0; shoot dry weigh (SDW), 12.7 mg; root dry weight (RDW), 10.2 mg; total biomass (TB), 18.1 mg.

f e r e n c e in t h e b i o m a s s a c c u m u l a t e d in e i t h e r t h e h e a t to le r -

a n t DTO-33, DTO-28, DTO-2, LT-7, LT-6, D~sir~e, NDD277-2,

a n d Y84-027, t e m p e r a t e a d a p t e d R u s s e t B u r b a n k , o r t h e h e a t

s e n s i t i v e 11401-01 a n d 9120-05. B a c t e r i z a t i o n a t 20 /15 C

i m p r o v e d to ta l b i o m a s s a c c u m u l a t i o n in 7 c lones , Maine-47,

LT-1, LT-9, D~sir~e, DTO-2, DTO-28, a n d 11401-01 as c o m -

p a r e d to t he n o n - b a c t e r i z e d con t ro l s , w h e r e a s b a c t e r i z a t i o n

a t 33/25 C i n c r e a s e d to ta l b i o m a s s a c c u m u l a t i o n in 15 c l o n e s

(Tab le 1). Bac t e r i zed LT-8 a c c u m u l a t e d t h e h i g h e s t b i o m a s s ,

f o l l o w e d b y t he b a c t e r i z e d DTO-2, Maine-47, a n d LT-5.

A l t h o u g h t h e r e w e r e c l o n a l d i f f e r ences in r e s p o n s e to

33/25 C in vitro, s e p a r a t i o n o f h e a t t o l e r a n t a n d s e n s i t i v e

c l o n e s w a s n o t poss ib le .

Ex vitro Tuber iza t ion

A l t h o u g h t e m p e r a t u r e s t r e s s (33 /25C) c a u s e d s e v e r e

r e d u c t i o n in t u b e r n u m b e r a n d t u b e r f r e s h weight , h ighly sig-

n i f i can t (P=0.0001) t e m p e r a t u r e X c l o n e i n t e r a c t i o n s w e r e

r e c o r d e d for b o t h var iables . A s ign i f ican t (P=0.0365) t empe r -

a tu re X b a c t e r i z a t i o n i n t e r ac t i on fo r t u b e r n u m b e r w a s a lso

found . I n d e p e n d e n t l y o f bac t e r i za t ion , a t 33/25C, ABA-defi-

c i en t c lones d id n o t p r o d u c e a n y t u b e r s a n d c lones LT-8, LT-

9, Y84-027 a n d DTO-28 t u b e r i z e d v e r y p o o r l y ( T a b l e 2).

DTO-33, D~sir~e, LT-1, a n d K e n n e b e c gave t he h ighes t h u m -


E x vi tro tuberization responses (tubers per pot ) o f bacterized and non-bacterized potato clones, Kennebec (A) , LT-2 (B), and LT-7 (C) at 20/15 and 33/25 C growth temperatures.

ber of tubers per pot. D~sir~e produced more tubers at 33/25

C than at 20/15C, but of a lesser weight. Tuber weight was the highest in Kennebec, followed by LT-1, D~sir~e and LT-7. Nei- ther tuber number in DTO-33, LTb2, Kennebec, DTO-28 and

Russet Burbank, nor tuber fresh weight in DTO-2 and LT-1 were significantly reduced by the high temperature as compared to 20/15 C (Table 2). There was no significant effect of bacterization on tuberization responses at 20/15 C. Under the

heat stress, however, bacterized treatment gave significantly (P=0.0126) more tubers and of a higher total fresh weight than the non-bacterized LT-7 (Table 2 and Fig. 3) and significantly

(P=0.0037) reduced total tuber weight in DTO-2 (Table 2).

DISCUSSION

As anticipated, bacterization modified i n vitro perfor-

mance of potato plantlets. The observed enhancement of bio-

mass in bacterized plantlets could be explained by the stimulation of root branching and root hair formation (Frommel

et al. 1991) and consequently, a better nutrient and water uptake. The ability of the PsJN strain to increase medium pH (Nowak and Stevens 1996) and cytokinin content (Lazarovits

and Nowak, 1997) may have contributed to biomass alloca- tion to roots.

Three way interaction between temperature, bacterization and potato genotype indicates the importance of clonal selection for utilization of beneficial microorganisms in potato production under heat stress conditions. Although the tested clones responded differently to temperature and bacterization i n vitro, with regards to root growth in partic- ular, at higher temperature. Bacterial stimulation of the change in S/R biomass partitioning in favor of the roots may

1998 BENSALIM, eta/.: PERFORMANCE OF CLONES OF POTATO 151

TABLE 2. - -Temperature and bacterization effects on tuber

yield o f 18 clones o f potato at two dif ferent

temperatures.

Tuber Number Fresh weight (g)

Clone Trt 20/15 C 33/25 C 20/15 C 33/25 C

11401.011" C 21.3 0.0 34.7 0.0 B 19.7 0.0 16.7 0.0

9120-05 1" C 5.0 0.0 7.9 0.O B 8.0 0.0 15.2 0.0

LT-1 C 27.3 13.0 158.9 119.3 B 18.0 17.3 136.9 109.9

LT-2 C 16.0 11.0 122.9 62.1 B 14.3 9.7 120.1 86.2

LT-5 C 29.7 16.0 199.5 102.4 B 22.3 8.3 174.1 89.9

LT-6 C 23.3 13.0 182.6 76.6 B 21.0 J 13.0 168.2 71.0

LT-7 C 16.0 3.0 184.2 92.6 B 23.0 17.3 175.2 157.2

LT-8 C 28.3 4.7 185.7 50.9 B 16.3 4.7 148.4 33.8

LT-9 C 26.7 3.7 179.4 45.2 B 23.7 6.3 165.9 63.4

DTO-2 C 32.7 11.3 184.0 136.7 B 35.7 13.0 224.2 76.5

DTO-28 C 14.3 8.0 209.2 75.5 B 12.0 5.7 187.9 73.8

DTO-33 C 30.3 21.4 139.7 87.2 B 31.2 22.4 162.8 99.7

Kennebec C 16.7 10.3 175.0 120.6 B 15.7 16.3 197.0 150.6

R.Burbank C 14.7 7.3 154.6 81.2 B 12.3 10.3 162.8 78.8

Maine-47 C 14.7 7.7 148.2 76.5 B 10.7 10.7 138.9 72.7

Ddsirde C 14.0 19.7 116.5 94.8 B 20.7 18.0 165.4 94.2

NDD277-2 C 21.7 11.0 135.8 64.0 B 18.3 13.0 146.1 76.7

Y84-027 C 19.7 4.7 198.3 34.8 B 20.0 4.3 242.6 62.0

Values per plant are means of 3 replicates. LSD 05 values: Tuber number, 9.2; Tuber fresh weight, 51.9 g. Treatment (Trt): non-bacterized (C); bacterized (B) plantlets. Heat acclimated and non-acclimated 4 week-old plants were potted for 12 weeks. * excluded from ANOVA (See text for explanation).

allow the plant to endure heat and water stress and eventu-

ally lead to greater yields. Indeed, our field and greenhouse

experiments confirmed better survival and performance of

bacterized plantlets compared to non-bacterized controls

when transplanted under water or heat stress (Nowak et al.

1995). Bacterization may also have practical implications for

t issue culture laboratories. Bacterized plant le ts no t only

grow faster but are sturdier, with better developed root mass

and produce more vigorous transplants (Nowak et al. 1995,

Nowak et al. 1997), capable of withstanding low fungal dis-

ease pressure significantly better than non-bacterized ones

(Nowak et al. 1997, Sharma and Nowak 1998). Moreover, the

bacterium establishes endophytic and epiphytic populations

in tissue culture plantlets (Frommel et al. 1991, Pillay and

Nowak 1997) and thus there is no need for re-inoculation if

the plantlets are multiplied by nodal explants.

The ABA-deficient mutants, 11401-01 and 9120-05, had

spindly appearance with small leaves both i n vitro and ex

vitro. They both benefitted from bacterization in vitro (Table

1). The bacterium induced shoot growth promotion in 11401-

01 at 20/15 C was so dramatic, that we think this clone has

potential for utilization in bioassays for plant growth pro-

moting ability of microorganisms. Despite such benefits i n

vitro, the transplants of the ABA-deficient mutants did not

benef i t f rom bacter izat ion ex v i t ro (Table 2). At 20/15 C

mutants produced small tubers with significantly lower f~esh

weight than the other clones, and at 33/25 C they both died 4

weeks after transplanting, independent ly of bacterization.

Plants of the ABA-deficient mutants are similar to the droopy

potato plants, which had high stomatal conductance due to

the very little ABA accumulation (Quarrie, 1982). This con-

firms the crucial role of abscissic acid in heat stress survival.

Our earlier study of the bacterization effect on i n vitro tuber-

ization responses in potato indica ted that the bac te r ium

induces synthesis of a tuberization factor, presumably ajas-

monate (a similar stimulation was achieved with jasmonic

acid) (Nowak et al. unpublished). The assumption was that

this compound may compensate for the ABA deficiency in

the mutants. The observed acceleration of tuberization (Fig.

3) and senescence in bacterized LT-7 plants (Bensalim 1997)

further supported this assumption.

Our data show that some clones originally selected for

heat stress tolerance, e.g. LT-1, LT-5, LT-6, LT-7, LT-8, LT-9,

DTO-2, and Y84-027, were more sensitive to high tempera-

ture than the temperate cultivars, Russet Burbank and Ken-

nebec (Table 2). The bacterization, which in greenhouse

experiments in fiats, enhanced stolon production and tuber

initiation (Dunbar 1997), under the heat stress significantly

increased tuber number in LT-7 (Table 2, Fig. 3). The non-

bacter ized LT-7 plants grown at 33/25 C produced m a n y

stolons but only a few tubers. On the contrary, stolon num-

ber in the bacterized LT-7 was reduced but the plants set

almost six times as many tubers. It is also interesting to note,

that in DTO-2 bacterization enhanced tuber fresh weight at

20/15 C and significantly reduced at 33/25 C (Table 2).


The expe r imen t s ind ica te tha t ut i l izat ion o f benef ic ia l

bac ter ia has a great po ten t ia l in t i ssue cul ture p lan t le t pro-

duct ion as wel l as the i r adapta t ion to hea t stress. To utilize

the benefits of bac te r iza t ion to its full potent ial , one should

consider genotypic se lec t ions (both, plant and microbia l ) for

spec i f ic env i ronments . Benef ic ia l b a c t e r i a c o u l d be intro-

duced into t issue cul ture o f po ta to at the early mul t ipl icat ion

s tages and p ropaga ted th rough success ive genera t ions . Our

r ecen t data also ind ica te t ha t bac te r i a inhabi t ing p lan t le t s

can t r ans loca te to s e e d t u b e r s ( N o w a k and S turz u n p u b -

l ished) and potent ia l ly benef i t the n e x t genera t ion o f po ta to

plants.

ACKNOWLEDGMENT

The authors w o u l d l ike to thank Dr. Henry de J o n g f rom

the Agr icu l tu re and A g r i - F o o d C a n a d a R e s e a r c h Sta t ion,

Fredericton, New Bnmswick , for his kind supply o f the ABA-

deficient mutants and for the f requent d iscuss ions dur ing the

tenure of this project , and Dr. Tess Astatkie f rom the Depart-

men t of Mathemat ics and Physics, N o v a Scot ia Agricul tural

College, Truro, Nova Scotia, for his ass is tance wi th ~ c s .

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