Biological Control of Plant Growth Using the Plant Growth-Promoting Rhizobacterium Bacillus mojavensis KJS-3

Bacillus mojavensis KJS-3를 이용한 식물의 생육촉진 활성연구

Abstract

Biological control using the plant growth-promoting Rhizobacterium (PGPR) has received significant attention in recent years. PGPR has been linked with promoting growth in economically important crops, such as potatoes, tomatoes, and rice. Bacillus mojavensis KJS-3 (Moja-3), isolated from food waste, possesses antifungal properties against Aspergillus terreus, A. fumagatus, A. flavus, and Fusarium redolense, and it may have potential in the development of products for industrial applications. The main purpose of this study was to determine the effects of spraying the PGPR Bacillus mojavensis KJS-3 on the growth of altari radish (leaf number, leaf length, leaf weight, root length, and rhizome length, adjacent portion diameter, and weight) and lettuce (leaf number, length, width, and weight). Three different concentrations of the foliar spray treatment of B. mojavensis KJS-3 were applied to the altari radish and lettuce: the recommended standard concentration of $1{\times}10^9cfu/g$, half the standard concentration of $0.5{\times}10^9cfu/g$, and double the standard concentration of $2{\times}10^9cfu/g$). The B. mojavensis strain foliar spray treatment increased the growth of the leaves and roots of the altari radish and increased the growth of the lettuce leaves. For both plants, the recommended concentration of B. mojavensis KJS-3 produced better growth than half the standard concentration, and the growth was similar with the double dose. This study demonstrates positive effects of Moja-3, suggesting it may be a potential new bio-fertilizer for improving the growth of altari radish and lettuce.

식물성장촉진 뿌리박테리아(PGPR)을 사용한 생물학적 조절(Biological control)은 최근 몇년 동안에 주목 받게 되었다. PGPR은 산업적으로 중요한 감자, 토마토 그리고 쌀과 같은 경제적으로 중요한 작물의 성장촉진과 관련되어있다. 음식물 쓰레기에서 발견된 Bacillus mojavensis KJS-3은 Aspergillus terreus, A. fumagatus, A. flavus and Fusarium redolense에 대해 항진균작용을 가지고 있는 균주로, 이미 여러 산업적인 측면에서 유용한 가능성이 확인된 균주이다. 본 연구에서는 Bacillus mojavensis KJS-3를 알타리무와 상추에 $0.5{\times}10^9cfu/g$, $1.0{\times}10^9cfu/g$ 및 $2.0{\times}10^9cfu/g$의 3가지 농도로 분무, 재배한 후, 잎의 수, 길이, 무게, 뿌리와 근경의 길이, 넓이 및 무게를 비교함으로써, 이들에 대한 생육촉진작용을 확인해보았다. 그 결과, 처리하지 않은 군에 비하여 Bacillus mojavensis KJS-3를 처리한 군이 더 높은 생육성장을 보였으며, $1.0{\times}10^92cfu/g$농도에서의 재배는 저농도에서의 재배보다 더 높은 생육 성장을 그리고 고농도에서의 재배와 비슷한 생육성장을 나타내었다. 이러한 결과를 토대로 B. mojavensis KJS-3의 생물학적 비료로서의 가능성을 확인할 수 있었으며, 또한 B. mojavensis KJS-3의 다른 작물에 대한 생육조절제의 적용도 가능할 것으로 예상된다.

Introduction

In modern agriculture field, increase in use of chemical pesticides and chemical fertilizers has resulted in deterioration of soil fertility and manifestation of pesticide-resistant mutants of insects and plant pathogens, ultimately causing harm to human body. To overcome these problems, biological control using PGPR has gained increased attention and much research works have been carried out in recent years. PGPR is a specific group of (bacterial and fungal) microorganisms that can be found located inside wide varieties of plant tissue types: fruit [30], seeds and ovules [23], stems [22], roots [13, 16] and tubers [15, 33]. Most of these PGPRs are members of common soil bacterial genera such as Bacillus, Pseudomonas and Burkholderia [21]. The PGPR has been found promoting growth in several essential crops such as wheat, soybean, lettuce, bean, maize, barley [19], potatoes [6, 12, 24, 33], tomato [20, 28], and tomato and cucumber [9]. Bacillus subtilis (Ehrenberg), the oldest gram-positive spore-forming bacterium, has proven to be safe as a non-pathogenic species and have been used enormously in various human food preparations for many years. The bacterium isolated from several botanical environments, mainly from the soil, has displayed properties similar to that of antibiotics with biological control potential. A strain of B. subtilis (RRC101) initially identified as Enterobacter cloacae and recently reported as a corn endophytic [14] is intercellular, nonpathogenic and growth enhancer, protecting the plants against fungi. Also, the report suggests that this strain can colonize plants from the seed application. This strain has been patented (Patent No. 5,994,117; ATCC 55732) as a biological control for treating the fungal infections in maize [4]. Subsequently, this isolate was found closely related B. subtilis like phenotype that was recently described as Bacillus mojavensis [27]. The mojavensis strain is a relatively new member of the genus Bacillus, and is known to be a PGPR of plants that can be distinguished from its closest relative Bacillus subtilis, with divergence only in their DNA sequences and fatty acid compositions [27]. The study of B. mojavensis appears to be profoundly valuable due to its effect on plant diseases and mycotoxins, possessing antifungal activity against pathogenic fungi in plants. Antifungal agent as a cyclic lipopeptide produced in Bacillus mojavensis was recently identified by Dr. Bacon [31]. Bacillus mojavensis presents additional qualities of being competitive with some fungi, mostly those that are also PGPR by the production of isomers of the surfactins, a biosurfactant that is described as being the most potent familiar of lipopeptide group. The surfactins are very active, readily degradable thus becoming more attractive for use in biocontrol [3]. On the other hand, Bacillus mojavensis KJS-3 (B. mojavensis KJS-3) was identified by Prof. Jae Seon Kang from the food wastes, and its antimicrobial activity was examined against some harmful fungi such as Aspergillus terreus, A. fumagatus, A. flavus and Fusarium redolense, adopting the spot-on-lawn method [17]. This strain has been patented as a biological control for diseases in human caused by fungi, MRSAs, and vancomycin-resistant Enterococci [18]. The aim of this study was to determine the biological control for the growth of altari radish and lettuce, by using PGPR Bacillus mojavensis KJS-3.

 

Materials and methods

Materials to study effects on growth of altari radish and lettuce

Bacterial strains, culture condition, media

The bacterial strain, B. mojavensis KJS-3 KCCM10961P was produced in Tryptic Soy Broth (TSB, Bacto) media and propagations generated in TSB on a rotating shaker (200 rpm/min) at 42℃. The formation of spores of B. mojavensis KJS-3 was done in medium containing corn starch 15 g/l, yeast extract 5 g/l, K2HPO4 2.5 g/l, KH2PO4 2.5 g/l, KCl 0.5 g/l, NaCl 0.3 g/l, MgSO4·7H2O 0.5 g/l, MnSO4·H2O 0.03 g/l and CaCl2·2H2O 0.2 g/l. Then, all the spores were cultivated under experimental conditions for 5-6 days at 42℃. All of the media utilized in this study were autoclaved at 121℃ for 15 minutes.

Plant site for cultivation

The cultivation of altari radish and lettuce were done in the farm of Dong-A University, Gimhea, South Korea and the effect of B. mojavensis KJS-3 was studied by comparing the growth of altari radish and lettuce.

B. mojavensis KJS-3 treatment method

Foliar spray treatment

100 g, 50 g and 200 g of B. mojavensis KJS-3 each were suspended separately in 20 liter water to prepare the three samples in the concentration of 1×109 CFU/g, 0.5×109 CFU/g and 2×109 CFU/g respectively. The standard concentration sample of 1×109 CFU/ g bacterial suspension was sprayed on day 7 for three times, when 3-4 leaves of altari radish were developed.

Treatment area

As shown in Table 1, twelve different plots (with total area of 60 m2) were allocated for altari radish as well as lettuce, separately. These plots were randomly treated with three different samples of Moja-3, for three times.

Table 1.Treatment area and test methods for altari radish and lettuce

Area layout for Moja-3 test in altari radish and lettuce

To evaluate Moja-3 effects, 12 testing areas were divided to control, half dose, standard dose and double dose treatment. The areas are shown in Fig. 1.

Fig. 1.Test area layout for cultivation of altari radish and lettuce. 12 testing areas are divided to 4 sections for cultivation of altari radish and another 12 areas were tested for lettuce. Control, half dose, standard dose and double dose treatments were applied to each section and the divided areas are shown in Fig. 1.

Cultivation methods

For the cultivation of altari radish and lettuce, their seed were taken from Heungnong province and their specifications have been shown in Table 2.

Table 2.Overview of altari radish and lettuce

Planting, cultivation and harvesting of altari radish

We cultivated altari radish following to the timeline shown in Table 3.1.

Table 3.1.Timeline for altari radish cultivation and harvesting

Planting, cultivation, and harvesting of lettuce

Timeline for the lettuce cultivation is shown in Table 3.2.

Table 3.2.Timeline for altari radish cultivation and harvesting

 

Results and Discussion

Effect of Moja-3 foliar treatment on altari radish

Effect of Moja-3 foliar treatment on the characteristics of altari radish leaves

The results of Moja-3 foliar treatment (up to three weeks, once each week) on leaves of altari radish have been summarized in Table 4.1a below. After harvesting, the growth characteristics of three different groups of altari radish treated with half, standard and double concentration of Moja-3 were tested by comparing the number, length and weight of the leaves with untreated control group. The number of leaves of control group was 15.21, and those treated with half dose, standard dose and double dose were 16.84, 18.22 and 18.21 respectively. While, the length of leaves of control group was 57.17 cm, those treated with half dose, standard dose and double dose of Moja-3 foliar spray were 61.28 cm, 63.78 cm and 63.74 cm respectively. The weight of leaves of control group was 191.14 g and those treated with half dose, standard dose and double dose were 202.14 g, 225.28 g and 222.04 g. These results suggest that, compared to untreated control group, the Moja-3 foliar spray group showed an increase in growth of altari radish leaves. As there were no significant differences in the growth characteristics (number, length and weight) of altari radish leaves when treated with the recommended standard and double concentration of Moja-3, the standard dose of foliar spray treatment was the preferred one over half dose test sample.

Table 4.1a* Means in a column followed by same letter are not significantly different according to Duncan’s multiple range test at the 1% level (p≤0.01).

Table 4.1.b* Means in a column followed by same letter are not significantly different according to Duncan’s multiple range test at the 1% level (p≤0.01).

Effect of Moja-3 foliar treatment on the characteristics of altari radish roots

After harvesting the altari radish, roots of altari radish were compared between the untreated control group and 3 different foliar spray Moja 3 treatment groups. Length of the root of control group was found to be 13.35 cm whereas the length of altari radish treated with half concentration, standard concentration and double concentration of Moja-3 foliar spray group were 14.27 cm, 14.51 cm and 14.48 cm respectively. Rhizomes of the control group was 6.54 cm and the altari radish treated with half dose, standard dose and double dose of Moja-3 foliar spray group were 7.61 cm, 7.75 cm and 7.70 cm respectively. Similarly, the diameter of adjacent portion of control group of altari radish was compared with Moja 3 treated group and found higher (4.85 cm) in case of standard dose of Moja-3 treated group. Weight of the root of control group altari radish was 201.21 g, while those of half dose, standard dose and double dose were 226.85 g, 235.08 g and 233.18 g. The growth in weight of the altari radish roots due to standard dose was more acceptable; it was higher than the growth in weight of the roots due to half dose (226.85 g) and even similar to double dose (233.18 g). In the present work, the use of 1×109 CFU/g concentration of Moja-3 treatment group showed the higher impact on the growth of root of altari radish.

Bacillus mojavensis KJS-3 foliar spray treatment for the growth of altari radish

Result of Moja-3 foliar spray in the altari radish, for three weeks once each week, did not showed any inhibitory effect on the growth of altari radish, and the results observed have been shown in Fig. 2 below.

Fig. 2.Altari radish during and/or after Bacillus mojavensis KJS-3 treatment.Cultivation procedure and altari radish growth appearance after Bacillus mojavenis KJS-3 treatment (Foliar Spray) : 2.1-day 1 of Moja-3 treatment, 2.2-devastating, 2.3-growing, 2.4-before harvesting, 2.5-after harvesting, 2.6-comparison of study groups.

Effects of Moja-3 foliar treatment on the characteristics of the lettuce leaves

Result of Moja-3 foliar treatment on the lettuce leaves have been summarized in Table 4.2 below. After harvesting three different groups of lettuce treated with half, standard and double doses of Moja-3, growth characteristics were examined by comparing the quantity, length and weight of the leaves with the control group. The number of leaves of control group was 20.15, while those treated with half dose, standard dose and double dose were 22.14, 23.84 and 23.71 respectively. The length of control group leaves was 22.17 cm, while the length of the leaves treated with half dose, standard dose and double dose were 24.18 cm, 26.29 cm and 26.07 cm respectively. The width of the control group leaves was 13.51 cm while those treated with Moja-3 foliar spray half dose, standard dose and double dose were 15.02 cm, 16.14 cm and 16.08 cm respectively. The weight of control group leaves was 157.58 g and those treated with half dose, standard dose and double dose were 170.10 g, 174.86 and 170.44 g. The growth in weight of the lettuce leaves due to standard dose (174.86 g) was more acceptable; it gained higher growth compared to the growth from half dose (170.10 g) and double dose (170.44 g). These results suggest that, compared with untreated control group the Moja-3 foliar spray group showed an increase in growth of the leaves of lettuce, and among there different group standard foliar spray treatments group showed the good result.

Table 4.2.* Means in a column followed by same letter are not significantly different according to Duncan’s multiple range test at the 1% level (p≤0.01).

Bacillus mojavensis KJS-3 foliar spray treatment for the growth of lettuce

Result of Moja-3 foliar spray in the lettuce, for three weeks once each week, did not showed any inhibitory effect on the growth of lettuce, and the results observed have been shown in figure 3 below.

Fig. 3.Lettuce during and/or after Bacillus mojavensis KJS-3 treatment. Cultivation procedure and lettuce growth appearance after Bacillus mojavenis KJS-3 treatment (Foliar Spray) : 3.1-day 1 of Moja-3 treatment, 3.2-devastating, 3.3-growing 3.4-before harvesting, 3.5-after harvesting, 3.6-comparison of study groups.

This is the very first study on growth promoting effects of bacterial application in the growth parameters of plants such as altari radish and lettuce. Though, similar investigations were conducted in different plant species, their studies suggested that, bacterial applications including Pseudomonas and Bacillus strains can stimulate growth and increase the yield in pepper and tomato [20, 28], in beans [32], in sugar beet [5], in spring barley [29], in sweet cherry [10], apricot [1], in raspberry [25] in apple [2], in rocket [8] and in tomato and cucumber [9]. The key reason for the growth promoting effect of bacterial applications on plant growth is that they affect on fixation capacity of Nitrogen [7, 26, 34] and is one of the most prominent mechanism of action with high probability of affecting plant growth [10].

In modern agriculture field, increase in use of chemical pesticides and chemical fertilizers has resulted in deterioration of soil fertility and manifestation of pesticide-resistant mutants of insects and plant pathogens damaging human body. To overcome these problems, biological control using endophyte is becoming more and more attentive and much research has been carried out in recent years. The species mojavensis is a relatively new member of the genus Bacillus and is known to be a PGPR of plants. Bacillus mojavensis KJS-3 discovered from food waste has been investigated as potential for developing the product in the industrial applications and found to possess antifungal property against Aspergillus terreus, A. fumagatus, A. flavus and Fusarium redolense. The effects produced by three different B. mojavensis strain foliar spray treatment method resulted increase in the growth of both altari radish and lettuce. The standard recommended dose of B. mojavensis KJS-3 (1X109 CFU/g) was found to be more effective than half of the dose and similar to the double dose, for the growth of both altari radish and lettuce. The present study demonstrates a significant positive effect of B. mojavensis KJS-3 as a potential agent of new bio-fertilizer for growth of altari radish and lettuce cultivation.