The long-term colonization dynamics of endophytic bacteria in cucumber plants, and their effects on yield, fruit quality and Angular Leaf Spot Disease

https://doi.org/10.1016/j.scienta.2021.110005Get rights and content

Highlights

  • Both Endophytic bacteria (EB) isolates survived in plant organs up to the 62 days.

  • The EB population density decreased from 105 to 103 CFU g plant−1 with plant age.

  • EB increased the yield by 22% and 21% with CB36/1 and CC37/2, respectively.

  • The EB strain CC37/2 reduced the disease by 41% and prevented the yield losses.

Abstract

Endophytic bacteria (EB) are ubiquitous in most plant species and they colonize plant tissues actively and systemically. Their colonization and persistence in the plant may be crucial to plant growth, yield and suppressing diseases. In this study, the long-term population dynamics of the two EB, namely Ochrobactrum spp. strain CB36/1 and Pantoea agglomerans strain CC37/2 in cucumber plants (Cucumis sativus L. cv. Gordion F1), were monitored. Also, the potential effects of these EB on plant growth, fruit quality, and yield, as well as the influences on Angular leaf spot disease of cucumber (ALS) and the yield under the disease pressure, were investigated. Plants were grown using the soilless cultivation technique in the greenhouse. Inoculation of the EB took place twice, and their population in plant tissues were monitored periodically for 62 days. Both EB isolates survived in plant organs until the end of the growing season, but over time, population densities dropped from 105 to 103 CFU g. plant−1 with the age of the plant. The EB applications had significant effects on fruit length, color and firmness. Total yield increased by 22% with CB36/1 and 21% with CC37/2 without disease pressure. Only CC37 / 2 significantly reduced the severity of ALS disease by 41% and increased yield by 22% compared to pathogen treatment alone. In this study, it was observed that the EB strain CC37/2 might contribute to reducing the chemical input and prevent the yield losses in soilless growing systems within the integrated agricultural concept.

Introduction

Cucumbers are attacked by several insects and affected by several pathogens. These pests and diseases may cause qualitative and quantitative damages in the cucumber plant. Angular leaf spot disease (ALS) that arises from Pseudomonas syringae pv. lachrymans (Psl) is the most common bacterial disease in cucumbers globally (CABI, 2016). ASL may reduce the cucumber yield by 30–60% (Pohronezny et al., 1978; Khalif, 1995).

The ALS presents symptoms in all cucumber plant organs except the roots. Systemic infection of the cucumber stems, leaves, fruits and seeds by pathogenic Psl is making it difficult to control the disease. Moreover, P. syringae pathovars may epiphytically grow on plant surfaces and build up a constant inoculum for infection (Hirano and Upper, 2000). Management of the disease by chemicals is the most common method. Several studies reported the development of resistance against streptomycin and copper that are commonly used chemicals for the control of Psl and other P. syringae pathovars (Yano et al., 1978; Scheck et al., 1996; Vidaver, 2002). Therefore, pesticide use does not always achieve the desired results. In addition, the adverse effects of the pesticide use on the environment and human health are well understood recently (McManus et al., 2002). The importance of biological control of plant diseases, especially the use of Endophytic bacteria (EB), one of the most promising biological control agents, is increasing day by day (Bruce, 2010).

EB are natural members of the group of Plant Growth-Promoting Rhizobacteria (PGPR or PGPB). The permeation and establishment of the EB in the tissues and protection of their density are essential for their contribution to the plant.

Common bacterial endophytes originate from the rhizosphere, which attract microorganisms due to the presence of root exudates (Hardoim et al., 2015). Mercado-Blanco and Prieto (2012) suggested that the entry of bacterial endophytes into roots occurs via colonization of root hairs.

EB enter the plants through cracks during the root growth and lateral root development (Rosenblueth and Martínez-Romero, 2006). They settle into the intercellular spaces, parenchyma, and cortical cells in the roots; they move into the xylem tissues, and in this way, colonizing leaves and stems (Ryan et al., 2008). A specific host-bacteria interaction is required to proceed from the rhizoplane to the root cortex and into the endogenous tissues because bacteria that reach the root cortical zone are blocked by endodermis, and very few bacteria could overcome this barrier (Compant et al., 2010).

EB have some advantages over epiphytic PGPR, although they contribute to plant health and development with mechanisms similar to other PGPR. In general, PGPB or EB can directly or indirectly affect plant growth and development (Saharan and Nehra, 2011). PGPB directly contributes to the plant by producing plant growth hormones (van Loon, 2007), by reducing the level of stress-induced ethylene in the plant through 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) production (Glick et al., 2007), by transforming nutritional elements into a form that can be used by the plant and by stimulating the plant resistance mechanisms (Antoun and Prévost, 2006; Saharan and Nehra, 2011). They can also contribute to the plant growth indirectly by acting as a biocontrol agent (stimulating antagonist, competition or defence systems), promoting beneficial symbiotic associations or neutralizing xenobiotics (Antoun and Prévost, 2006; Saharan and Nehra, 2011). In addition to the mechanisms as mentioned above, EB, unlike epiphytic PGPB, can establish a closer association with the plant because they live in plant tissues, and the metabolites they produce can be directly perceived by the plant (Rosenblueth and Martínez-Romero, 2006; Hardoim et al., 2008). Since their colonization is not limited to a specific region in the plant (such as rhizosphere), they can be transported to other tissues using transmission systems allows them to combat the pathogens in all areas and through several mechanisms (Rosenblueth and Martínez-Romero, 2006; Hardoim et al., 2008). On the other hand, EB colonization in plants can help protect them from biotic and abiotic factors that limit their development in the exogenous environment and sustain their viability for a long time (Rosenblueth and Martínez-Romero, 2006; Mercado-Blanco and Lugtenberg, 2014).

It was reported that EB had an inhibitory effect with different levels against several fungal, oomycete (Cho et al., 2003; Chakraborty et al., 2009; Hassan et al., 2014; Larran et al., 2016) and bacterial diseases (Hahm et al., 2012; Yi et al., 2013; Akköprü et al., 2018) using one or more mechanisms mentioned above.

The endophytic bacteria trigger induced systemic resistance in tomato and pepper plants against P. syringae pv. tomato and Xanthomonas axonopodis pv. vesicatoria, respectively (Yi et al., 2013; Fujita et al., 2017). Akbaba and Özaktan (2018) reported that two EB isolates decreased disease severity caused by Psl on cucumber plants. However, only one of the isolates decreased the Psl population density simultaneously with the severity of the disease. The disease severity suppression without a decrease in pathogen population density might be explained by inducing systemic tolerance in the host plants (van Loon, 2007; Akköprü and Özaktan, 2018).

The bacterial endophytes can decrease the pathogen population by producing toxins or bioactive antimicrobial compounds (Pageni et al., 2014). It was shown that some bacterial endophytes could prevent cellular growth or metabolic activity of microbial pathogens by synthesizing certain lipopeptide molecules (Zhang et al., 2019). Siderophores produced by endophytic bacterial strains may be involved in the suppression of pathogens (Johnson et al., 2013). Furthermore, the siderophore production of EB was shown to play an important role in symbiosis with plants (Araújo et al., 2008). In addition to the direct contribution of IAA and ACC deaminase produced by EB to plant development, it was found that they also increase the chance of colonization in plants (Khalid et al., 2004; Etesami et al., 2015). IAA production enables the interaction of bacteria with plants (Etesami et al., 2015), and it may play a role in entering of the bacteria into endophytic life form in the plant (Verma et al., 2001). As in some of the examples mentioned above, endophytes can contribute to the plant in different ways by producing secondary metabolites, toxins, structural compounds, enzymes, antibiotics, siderophores, phytohormone and by reducing reactive oxygen species damage (Hardoim et al., 2015; Zhang et al., 2019).

It was reported that EB application increased leaf, shoot and root growth and yield in several vegetables, such as tomato, pepper, potato, and corn (Sturz et al., 1998; Kokalis-Burella et al., 2002; Armada et al., 2015; Ajilogba and Babalola, 2016). EB application significantly increased the levels of plant nutrients, such as potassium, calcium, copper, manganese, and rubidium, which affect plant development, yield and quality (Ajilogba and Babalola, 2016). In addition to the significant increase in crop production, several studies showed the positive effects of bacteria on food quality, such as improved vitamin, flavonoid and antioxidant content, among other benefits (Jiménez-Gómez et al., 2017). Also, it was found that PGPR application affected the amino acid, sugar, and volatile composition in ripe fruits, contributing to a more pleasant-tasting fruit without forfeiting selected quality indicators in tomato (Berger et al., 2017). Garcia-Seco et al. (2015) showed that PGPR application improved the fruit quality by triggering flavonoid biosynthesis in plants, which indicated some of the genetic targets of elicitation by beneficial bacteria. EB have protective potential against diseases parallel to their contribution to plant growth (Muthukumar et al., 2017).

The EB applications can positively contribute to plant health, and also increased yield with or without under stress. The success of EB is depended on their ability to survive and maintain the populations in the plants. More knowledge is needed about increasing their application at the commercial level. This study aimed to investigate the plant growth-promoting and biocontrol effects of two EB; Ochrobactrum spp. strain CB36/1 and P. agglomerans strain CC37/2, on cucumber plants. Under the study's frame, three experiments were conducted to monitor these EB's long-term population dynamics in cucumber plants, determine their effects on plant growth, yield and fruit quality, and test their ability to control ALS disease caused by Psl.

Section snippets

Tested plant material and endophytic bacteria

Two EB strains, Ochrobactrum spp. CB36/1 and P. agglomerans CC37/2, isolated from cucumber plant stems and leaves in a previous study, were selected for the present study (Özaktan et al., 2015). The Gordion F1 cucumber (Cucumis sativus L.) variety was used as plant material in the study.

EB inoculation

EB were inoculated to the plants using two methods which are seed coating and drenching: i) Seed coating: A suspension in 108 CFU/mL concentration was prepared with EB cultured for 48 h in King’s medium B

Monitoring the EB in the plant

The findings showed that EB CB36/1 and CC37/2 were adherent on seeds at 1.68x109 and 8.25x108 CFU/g. seed−1 concentration two hours after the application, respectively. At the cotyledons emergence stage (T1), the second EB application was conducted. It was observed that the colonization density reached high levels up to 1011 and 1010 CFU/g. plant levels in root and stem for both isolates. In the following periods, these levels remained at 105-104 CFU/g. plant levels, although fluctuations were

Discussion

Maintaining an adequate endophytic bacteria population in the plant is a key factor for EB to improve plant health or to exhibit their antagonistic effects against pathogens. Although the population of CB36/1 and CC37/2 isolates used in the present study reached 1011 CFU g. fresh plant−1 level after the second application, it decreased rapidly in a short period and generally remained at 105-104 CFU g. fresh plant−1 levels (Fig. 1). The population density of endophytes is variable. It may change

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Ahmet Akköprü: Methodology, Writing - original draft, Writing - review & editing. Şahika Akat: . Hatice Özaktan: Methodology, Writing - review & editing. Ayşe Gül: Methodology, Writing - review & editing. Mustafa Akbaba: .

Acknowledgements

The present study was sponsored by national grants from the Scientific and Technological Research Council of Turkey (TUBITAK-COST 111 O 505) and Ege University Science-Technology Research and Application Center (EBILTEM). The authors express their gratitude for the contribution of the participants in EU-COST Action FA1103.

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