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Screening for potential phytotoxin-producing streptomycetes isolated from soils in Jordan

Sereen M. Bataineh, Ismail Saadoun and Khalid M.Hameed

Dept of Applied Biological Sciences, JUST, P. O. Box 3030 Irbid 22110, Jordan, Email sereen_b@yahoo.com
isaadoun@just.edu.jo; hameed@just.edu.jo

Abstract

Twenty one active streptomycetes isolates out of 231 were recovered from 16 different regions in Jordan. The isolate R9 was most effective against the germination of cucumber and ryegrass seeds and their seedling growth, placed adjacent to 3 weeks old strip R9 culture on starch-casein-nitrate (SCN) agar plate. It caused 85.2% inhibition in seed germination of ryegrass and suppressed the growth of cucumber radicle (85%) and shoot (90.3%). R9 culture filtrate from glucose-peptone-molasses broth (GPM) caused complete inhibition of ryegrass seed germination. It caused 87% and 90.5% reduction in radicle and shoot growth of cucumber, respectively. In case of milk thistle there was 89% reduction in radicle growth only. Diluted R9 culture filtrate (1:1) caused complete inhibition of seed germination of redroot pigweed. Dichloromethane fraction of R9 culture filtrate 3 and 5 mg concentrations caused complete inhibition of ryegrass seed germination and severely reduced the seedling growth of cucumber and milk thistle.

Media Summary

Streptomycete isolates with phytotoxic activity were recovered from Jordanian soils. Further identification of the most active isolate and the active moiety involved is being undertaken.

Key Words

Streptomycetes, bioherbicides, cucumber, ryegrass.

Introduction

There are about 300 common weed species that cause crop losses world wide (Hoagland 1990). Chemical herbicides are widely used despite the growing public concerns of some undesirable consequences of using agrochemicals such as accumulation, biomagnifications and excessive persistence (Heisey and Putnam 1990). Furthermore, the intensive and arbitrary usage of herbicides leads to the development of resistant weed species to some of those herbicides (Mallik 2001). The augmentation in herbicidal use is due to reduction in tillage and the low agricultural input (Mallik 2001). The destructive impact on the environment from the continued use or misuse of agrochemicals justifies the search for biodegradable, selective, and eco-friendly herbicides (Hoagland 1990). Natural products such as secondary metabolites of certain microorganisms have been investigated as biological weed control agents. Potential use of microbial metabolites as bioherbicides became the alternative for agrochemicals (Mallik 1997). Soil microorganisms, in particular, have the capability to produce compounds that are potentially toxic to higher plants (Heisey et al. 1985). One group of those microorganisms was the actinomycetes and particularly streptomycetes for their potential to produce many extracellular active compounds. Many of those compounds were demonstrated to be bioherbicides such as anisomycin, bialaphos, herbicidans A and B (Mallik 2001).

Materials and Methods

Isolation of streptomycetes

Indigenous streptomycetes were isolated from soils in Jordan (16 locations) using spread plate according to Mallik (1997) using SCN agar plates instead of glucose-asparagine-dipotassium phosphate (GAP) agar and incubated at 28°C for 10 days under aerobic conditions. Streptomycetes colonies were recognized according to (Williams et al. 1972) and were maintained in pure slant culture according to Mallik (2001). The most active isolate R9 was preserved in 10% glycerol at -20°C recommended by Wellington and Williams (1978) for long time preservation of actinomycetes with minimal effect on their bioactivities and little impact on the genetic material.

Screening of streptomycetes isolates for their bioherbicidal activity

The strip cultures for streptomycetes isolates were prepared on SCN agar plates according to Mallik (1997). Inoculated agar plates were incubated at 28°C for 3 weeks to give sufficient time for streptomycetes growth and diffusion of metabolites in the medium (Mallik 2001). Uninoculated SCN agar plates were similarly incubated to serve as control. Surface sterilized cucumber (2% hypochlorate) and ryegrass (25% hypochlorate) seeds (6 of each per plate) were placed on two opposite sides of the culture strip and uninoculated control plates. Plates were incubated in dark at 28°C for 4 days. Seed germination and length of radicles and shoots were monitored.

Phytotoxin(s) secretion in the submerged culture

Primary inoculum of R9 was prepared according to Mallik (1997) and used to inoculate three different broth media , SCN, GPM and peptone-molasses-corn steep (PMC) inside 100 ml Erlenmeyer flasks (25 ml per flask) incubated at 28°C inside orbital shaker for 5 days with shaking speed of 140 rpm. Culture filtrates were used in bioassays for the phytotoxic activity.

Phytotoxic activity in the culture filtrates

This part was performed as described by Mallik (1997) using 2.5 ml of R9 crude culture filtrates from SCN, GPM and PMC broth moisten the filter paper placed inside Petri-dishes with seeds scattered on surface. Seeds (4) of monocotyledonous (ryegrass) and two dicotyledonous (cucumber, milk thistle) species were used in this bioassay. Controls treatments included uninoculated SCN, GPM and PMC broth alone (Halleck et al. 1955) and sterilized distilled water. The plates for each treatment were placed inside plastic bag in order to conserve moisture, and they were incubated at 28°C in darkness for 4 days. After that, the length of the radicles and shoots was measured and germination percentages were calculated. R9 culture filtrate from the GPM submerged culture diluted (1:1) with sterilized distilled water and tested against seeds of Amaranthus retroflexus.

Extraction of the phytotoxin(s) containing fraction

Submerged GPM culture (500 ml / 2L Erlenmeyer flask) of R9, incubated at 28°C for 7 days, shaken at 140 rpm were extracted with dichloromethane (1:3 V/V) in order to separate the phytotoxin(s) containing fraction (Mallik 1997). This fraction was brought to close dryness inside rotary evaporator at 29°C. The residue was recovered from the evaporator with 4 ml of dichloromethane and its exact dry weight was figured using jet stream of N2 gas. 50 mg of the resultant residue was reconstituted in 10 ml dichloromethane and used as stock concentration to prepare 1.5, 3 and 5 mg concentrations applied on filter papers inside glass Petri dishes and used in the bioassay after the solvent has completely evaporated. Seeds (3) of cucumber, ryegrass and milk thistle were placed over the filter papers and moistened with 2.5 ml sterilized distilled water, in three replicate plates. All of the moisture germination dishes prepared as described were placed inside plastic bags to conserve moisture and incubated for 4 days at 28°C. Then length of the radicles and shoots was measured and the germination percentages were calculated.

All experiments were performed in completely randomized design (CRD) and results were statistically analyzed (SAS Institute, 1999). Means were separated by the least significant differences (LSD) at α = 0.05.

Results

Screening for isolates with bioherbicidal activity

Out of the 231 isolates that were screened for phytotoxin production on agar plates (Figure 1) twenty one isolates (9.1%) showed such activity. This percent falls within the range of previous published results in cucumber, barnyardgrass, and cress where it was reported as 6.7% and 10-12%, respectively, (DeFrank and Putnam 1985; Heisey et al. 1985). The isolate R9 caused 85.2% inhibition in seed germination, 98.1% reduction in radicle growth and 97.1% reduction in growth of the shoot of ryegrass. R9, though it did not affect the germination of cucumber seed, but it caused 85% reduction in radicle growth and 90.3% reduction in shoot growth (Table 1).

Table1. Effect of R9 on seed germination, radicle and shoot growth of cucumber and ryegrass assessed by the agar plate screening method. *For the listed data selected from other columns in original table, not shown.

 

Control

R9

LSD*

Cucumber

     

% Germ.

100

100

0

% P.D.Germ.

 

0

 

R.L (mm)

59.3

8.9

7.5

% P.D.RL

 

85

 

S.L (mm)

27.7

2.7

9.7

% P.D.SL

 

90.3

 

Ryegrass

     

% Germ.

100

14.8

22.6

% P.D.Germ.

 

85.2

 

R.L (mm)

10.4

0.2

1.4

% P.D.RL

 

98.1

 

S.L (mm)

10.3

0.3

2.2

% P.D.SL

 

97.1

 

Figure 1. Screening for bioherbicidal activity by R9 strip culture technique. A: Control (SCN agar alone) with cucumber (left) and ryegrass (right) seeds. B: R9 culture with cucumber(right) and ryegrass (left) seeds.

Figure 3. Phytotoxic activity of the dichloromethane extracted fraction of R9 culture filtrate against cucumber, ryegrass and milk thistle seeds. A: Control (sterilized distilled water). B: 5 mg concentration of R9 extracted fraction.

Phytotoxic activity of R9 culture filtrates from SCN, GPM and PMC broths

SCN culture filtrate of R9 caused 33.3% inhibition in the seed germination of ryegrass compared to the SCN broth control (Figure 2) and 73.6%, 67.8% reduction in the growth of the radicle for cucumber and ryegrass, respectively. It significantly reduced the growth of the shoot for cucumber (96.1%), ryegrass (57.3%) and milk thistle (36.2%). GPM culture filtrate of R9 caused complete inhibition in seed germination of ryegrass (Figure 2).The effect of the R9 culture filtrate on ryegrass substantiates the finding reported by Mallik (1997). Germinated cucumber showed 87% and 90.5% reduction in radicle and shoot growth, respectively, while germinated milk thistle showed 89.1% reduction in the growth of the radicle compared to the GPM broth control. The same filtrate diluted 1:1 with sterilized distilled water caused complete inhibition to Amaranthus retroflexus seeds. The phytotoxic activity of R9 culture filtrate from its GPM broth culture disclosed here presents a pioneering step in Jordan toward a broad spectrum phytotoxin that affect both dicotyledonous and monocotyledonous plant seeds such as cucumber, ryegrass, milk thistle, and redroot pigweed in pre-emergence applications. PMC culture filtrate of R9 caused 74.1%, 78.6% reduction in growth of the radicle and shoot of cucumber, respectively, compared to the PMC broth control (Figure 2).

Extraction of the phytotoxin(s)

Dichloromethane extracted fraction of the R9 culture filtrate from GPM broth culture caused complete inhibition of ryegrass seed germination at 3 and 5 mg concentration (Figure 3), at 1.5 mg concentration, there was no inhibition of seed germination (Table 2). However, the radicle and shoot growth was reduced by 97.5% and 90.4%, respectively, compared to sterilized distilled water control. In case of cucumber, there was no inhibition of seed germination, whereas growth of the radicle was reduced by 95.6%, 98.1% and 99.4% at 1.5, 3 and 5 mg concentrations, respectively. The growth of cucumber shoot was completely suppressed at 3 and 5 mg concentrations and reduced by 97.7% at 1.5 mg concentration. Seed germination of milk thistle was significantly inhibited by 55.7% at 5 mg concentration only. Radicle growth of milk thistle was reduced by 66.5%, 93.2% and 96.6% at 1.5, 3 and 5 mg concentrations respectively, whereas growth of shoot was completely suppressed at 5 mg concentration, and significantly reduced by 73% at 3 mg concentration only. This activity on cucumber seeds coincide with the phytotoxic activity on cucumber apical growth done by Mishera et al. (1987). The phytotoxic activity observed here indicates that it may be a function of concentration coupled with the degree of susceptibility of each crop to the active ingredient produced.

Conclusion

This investigation though is a pioneering step in this direction, it indicated that cultivated soils, forests and barns in northern Jordan are good reservoirs for potential streptomycetes for phytotoxin production. The phytotoxic potential of R9 crude extract suggests a promising broad spectrum phytotoxin which needs advanced steps in identification.

Figure 2. Effect of R9 culture filtrate from its GPM, SCN and PMC broth cultures on germination, radicle and shoot growth of cucumber, ryegrass and milk thistle. Note: For the listed data selected from other original histogram, not shown. The bars represent the standard errors of the means for a species.

Table 2. Responses of cucumber, ryegrass and milk thistle on filter paper moistened with solvent extract of fresh R9 culture filtrate (CF) and uninoculated broth (UB). *Significantly different from corresponding uninoculated broth at α = 0.05

Concentration of extract
(mg/ml)

Radicle length (mm)

Shoot length (mm)

Germination (%)

UB

CF

UB

CF

UB

CF

Cucumber

           

1.5

75.0

3.7*

28.8

0.7*

100.0

100.0

3.0

73.0

1.6*

21.4

0.0*

100.0

89.0

5.0

55.8

0.5*

11.6

0.0*

100.0

89.0

control (S.D.H2O)

85.0

 

30.4

 

100.0

 

LSD

 

2.7

 

6.0

 

25.4

Ryegrass

           

1.5

17.7

0.4*

17.7

1.2*

100.0

55.7

3.0

14.8

0.0*

10.5

0.0*

100.0

0.0*

5.0

7.0

0.0*

2.4

0.0*

100.0

0.0*

control (S.D.H2O)

16.3

 

12.5

 

100.0

 

LSD

 

1.9

 

4.9

 

48.0

Milk thistle

           

1.5

31.5

7.9*

9.7

7.5

100.0

100.0

3.0

21.5

1.6*

7.1

2.4

100.0

66.7

5.0

20.0

0.8*

5.7

0.0*

100.0

44.3*

control (S.D.H2O)

23.6

 

8.9

 

100.0

 

LSD

 

4.7

 

3.5

 

36.6

Acknowledgements

The authors acknowledge Jordan University of Science and Technology for their financial support of this work. And acknowledge Prof. M.A.B. Mallik (Emeritus Research Professor, Langston University, Oklahoma, USA) for his guidance and valuable suggestions through out the present work.

References

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