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Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera co-cultured with plant growth-promoting rhizobacteria

Essaid Ait Barka, Abdel Belarbi, Cathy Hachet, Jerzy Nowak, Jean-Claude Audran
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09087.x 91-95 First published online: 1 May 2000


The potential of a plant growth-promoting rhizobacterium, Pseudomonas sp. (strain PsJN), to stimulate the growth and enhancement of the resistance of grapevine (Vitis vinifera L.) transplants to gray mould caused by Botrytis cinerea has been investigated. In vitro inoculation of grapevine plantlets induced a significant plant growth promotion which made them more hardy and vigorous when compared to non-inoculated plantlets. This ability increased upon transplanting. When grown together with B. cinerea, the causal agent of gray mould, significant differences of aggressiveness were observed between the inoculated and non-inoculated plants. The presence of bacteria was accompanied by an induction of plant resistance to the pathogen. The beneficial effect from this plant–microbe association is being postulated.

  • Vitis vinifera
  • Botrytis cinerea
  • Plant growth-promoting rhizobacterium
  • Pseudomonas

1 Introduction

Plant growth-promoting rhizobacteria (PGPR) are free-living bacteria having a beneficial effect on plants as they enhance emergence, colonize roots, and stimulate growth [1]. In recent years, the concept of PGPR for promotion of plant growth is gaining worldwide acceptance [2].

Endophytic bacteria may stimulate host plant growth through any of several possible mechanisms including biological control [3], induced systemic resistance to plant pathogens [4,5], phytohormone production and improvement of nutrient and water uptake [3,6]. Some of these organisms, bacteria and vascular–arbuscular mycorrhiza, can be utilized in agricultural and horticultural practices for the purpose of transplant protection against diseases, improvement of establishment and overall performance [7,8]. It may also improve plant performance in stress environments and consequently enhance yields [6,9]. Wei et al. [7] observed that some PGPR strains can induce systemic resistance in cucumber when grown together with Colletotrichum orbiculare.

The PsJN bacteria are capable of establishing endophytic and epiphytic populations, allowing clonal multiplication of plantlets by nodal explants in perpetuity, without the need for re-inoculation [9]. As endophytes, PsJN have the advantage of escaping microbial competition and also the host's response to pathogen attack [10].

Gray mould diseases caused by Botrytis cinerea are probably the most common and widely distributed diseases of vegetables, ornamental plants, fruits, and even field crops throughout the world. Chemical control of Botrytis has been partially successful. However, the risk of appearance and establishment of resistance to Botrytis is considerable.

In this study, a plant growth-promoting, non-fluorescent Pseudomonas sp. (strain PsJN) was chosen to test the hypothesis that the colonization of grapevine plants by the bacterium would enhance their development and growth and at the same time increase their resistance to attacks by B. cinerea. The objective of this study was to determine whether the presence of the bacterium would undermine the negative effects of B. cinerea; increase the growth and the development of grapevine plantlets; induce resistance against B. cinerea; and finally to observe whether the effect of the bacterium is transmitted to the next generation without re-inoculation.

2 Materials and methods

2.1 Plant material and in vitro growth conditions

Disease-free plantlets of Vitis vinifera L. cultivar ‘Chardonnay’ were obtained by growing nodal explants on Murashige and Skoog medium [11]. Vitro-plants were grown under 200 μE m−2 s−1 white fluorescent light, 16/8-h photoperiod, and 25°C day/night temperature. Multiplication of plants was conducted in 25-mm test tubes using 15 ml of medium per tube. For each experiment, 24 nodal explants were used.

2.2 In vitro inoculation

The bacterial inoculum was produced by transferring two loops of PsJN to 100 ml King's B liquid medium in a 250-ml Erlenmeyer flask and incubated at 20°C at 150 rpm for 48 h as previously described [12]. Bacteria were collected by centrifugation (3000×g, 15 min) and washed twice with phosphate saline buffer (PBS) (pH 6.5). The pellet was resuspended in the same PBS buffer, then used as inoculum [13]. About 1 cm long nodal explants, taken from 6-week-old plantlets, were immersed in the inoculum for 1 min, blotted with sterile filter paper, and transplanted into culture tubes. Non-inoculated controls were dipped in PBS only. The plants were grown in the growth chamber as above.

2.3 In vitro growth responses of the first and second plantlet generations after inoculation

Inoculated plantlets (first generation) were subcultured in vitro to obtain the second generation. Inoculation was carried out only on the initial explants, not on the second-generation explants.

2.4 Clonal responses to bacterial inoculation

Nodal explants taken from 6-week-old non-inoculated and inoculated plantlets were cultured on 15 ml medium in 25×200-mm test tubes. Growth responses were evaluated after 6 weeks in culture. Dry weights were determined after forced air-drying for 48 h at 70°C.

2.5 B. cinerea culture

B. cinerea (strain 630) was graciously provided by Dr. Brygoo (INRA, Paris, France). It was maintained on potato dextrose agar (PDA).

2.6 Evaluation of induced resistance

Six-week-old plantlets of the cultivar ‘Chardonnay’, both inoculated and non-inoculated ones, were grown with B. cinerea. Antagonism between B. cinerea and the PsJN bacteria was studied by placing both organisms on the same PDA plate and incubating them at 20°C for 7 days. A thin layer of PDA was removed aseptically and placed in a drop of sterile water on a glass slide. A coverslip was placed on the film, and the slide was observed under a microscope.

3 Results and discussion

It is known that the bacterium forms endophytic and epiphytic populations when co-cultured, either with potato [10] or with tomato [12]. The current study showed that grapevine plantlets co-cultured with bacteria grow faster and have significantly more secondary roots, root and leaf hairs (Fig. 1). These results were identical to those on potato [9], tomato [12] and other crops [10] which were inoculated with the same bacterium.


Effect of Pseudomonas strain PsJN on the growth of V. vinifera L. plantlets.

The present work indicates the possible ability of the PsJN bacterium to promote the growth of grapevine vitro-plants through two generations. Furthermore, the beneficial effect was more pronounced in the second generation when compared with the first. Comparable results have been observed on potato plantlets [9].

Shoot and root dry weight was the best indicator of the response of grapevine to inoculation with strain PsJN. Inoculation induced an enhancement of shoot and root dry weight as well as the number of nodes (Table 1), confirming the results obtained by Nowak et al. [9] with potato plantlets.

View this table:

Effect of bacterization with Pseudomonas sp. strain PsJN, on the in vitro growth response of grapevine plantlets cultivar ‘Chardonnay’

When B. cinerea was grown with the PsJN bacterium on the same PDA plate, a significant zone of inhibition was observed around the bacterial inoculum as shown in Fig. 2. Recently this type of inhibition has been shown using Pythium radiosum on B. cinerea[13]. In the zone of interaction with the PsJN bacterium, microscopic observation showed the presence of coagulation into cytoplasm of B. cinerea mycelium (Fig. 3b). In other cases, large vesicles appeared in cells of the mycelium (Fig. 3c), while others showed an empty mycelium devoid of cytoplasm after the inhibition by the bacterium (Fig. 3d). Similar observations were reported by Paul [13] in mycelium of B. cinerea co-cultured with P. radiosum.


Interaction between B. cinerea and Pseudomonas strain PsJN (center of the petri dish) when inoculated together on PDA.


Microscopic study of B. cinerea. a: Normal mycelium. b–d: Mycelium taken in the zone of interaction between B. cinerea and Pseudomonas strain PsJN. b: Partially emptied mycelium. c: Big vesicles in the mycelium. d: Emptied mycelium. Bar=40 μm (a), 100 μm (b–d).

Experiments with vitro-plants of V. vinifera L. cultivar ‘Chardonnay’ showed its susceptibility to fungal attack, producing the common symptoms of gray mould during the first week following the inoculation with B. cinerea. In contrast, under the same conditions inoculated vitro-plants were healthy, and exhibited only a small necrosis at the leaf surface in response to the pathogen attacks, thus confirming the result obtained in the bioassay. Similar observations were obtained with inoculation of potato plantlets with the PsJN bacterium co-cultured with Verticillium alboatrum[13], and Rhizoctonia solani[14]. Furthermore, PsJN applied in combination with Serratia plymuthica increased the resistance of tomato plants co-cultured with Fusarium oxysporum sp. lycopersici[15]. Other studies reported the effectiveness of inoculation with other PGPR strains as biocontrol agents [5,16,17]. Recently, the suppression of B. cinerea was obtained on grapevine plants co-cultured with P. radiosum[13].

Recent studies have shown that inoculated plants produce elevated levels of cytokinins, and may play a role in plant function [7,18]. As cytokinins are manufactured in the roots, the enhanced root branching might be an indication of better cytokinin supply. It has also been established that cytokinins play a central role not only in plant development, but also in the perception and transduction of environmental signals [19].

The experiments indicate that the utilization of beneficial bacteria has a great potential in the field of tissue culture as well as the adaptation of young plantlets to overcome pathogen attacks. As far as we know, this is the first time that such interaction between grapevines and the Pseudomonas sp. (strain PsJN) has been reported. However, the mechanism by which the PsJN bacterium stimulates the growth and development has not been investigated in this study. Production of phytohormones, siderophores, or other components could be involved. It has also been established that PsJN induces developmental [10] as well as biochemical [7,9] changes typical of the responses of induced systemic resistance [5,17].

Future work will elucidate the plant growth promotion mechanism(s) involved, and the role of PsJN in disease suppression. This strain may be used in the biocontrol of B. cinerea attacks as well as the improvement of the growth of grapevine plants.


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