Microbial Endophytes

Prospects for Sustainable Agriculture

Ajay Kumar,Vipin Kumar Singh

• Verlag: Woodhead Publishing
• Jahr: 2019
• ISBN:
  9780128187258
• Thema: Ciencias biológicas
• Thema: Botánica

Microbial Endophytes: Prospects for Sustainable Agriculture discusses the practical and theoretical aspects regarding the use of endophytic microorganisms in agriculture, providing insights on the biotechnological applications associated with long-term crop production. Chapters deal with the various aspects of endophytic microorganisms, including isolation, enumeration, characterization procedures, diversity analysis, and their role as biofertilizer, biocontrol agent and microbial inoculants. Framed to discuss the present and future potential of microbial endophytes in biotic and abiotic stress management, bioremediation, bioactive compounds production, and in nanotechnology, this book provides a single-volume resource that will be valuable to academics and researchers interested in microbiology, agricultural sciences and biotechnology.

• Explores aspects of sustainable agriculture by using endophytic microorganism such as bacteria, fungi and actinobacteria
• Presents insights into the use of endophytes as biofertilizer and biocontrol agents in sustainable agriculture
• Relates endophyte organisms and nano-technology

1

Isolation and characterization of endophytes: Biochemical and molecular approach

Dimitrios Manias^a,1; Akanksha Verma^b,1; Dharmendra Kumar Soni^{a,c a} Department of Molecular Biology, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden
^b Department of Botany, MLKPG College, Balrampur, India
^c Laboratory of Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden
¹ Both consider as first author.

Abstract

Endophytes are microorganisms residing inside the plants with an escalating interest for the scientific community. The plethora of endophyte-derived medicinal molecules and the application potentials in agriculture and bioremediation are some of the reasons why endophytes are extensively studied. Endophytes which can be either endophytic fungi or bacteria have been isolated from a wide variety of plants. The huge diversity among the endophytic microbes and the host plants hinders the establishment of a uniform protocol for isolation of endophytes. However, the most common technique that is currently used involves a thorough surface sterilization followed by fragmentation of the plant tissue and culture of the fragments onto agar plates. Nevertheless, non-culturable microorganisms are usually omitted from the aforementioned procedure and molecular approaches have to be applied for a more inclusive isolation. Sequencing of 16S rRNA for bacteria and ITS or 18S rRNA for fungi can be applied to both culturable and unculturable endophytes. Sometimes, morphological examination can be a further validation for the identification of the isolated strains. Subsequently, a variety of biochemical and molecular approaches including colorimetry, spot assays, chromatographies, and gene-targeted PCR (polymerase chain reaction) amplification are performed in order to further characterize the endophytic strains. Scrutinous functional analysis of endophytes may provide valuable insights into the advancement of agriculture, medicine, and industry. In this chapter, methods for the isolation of endophytic bacteria/fungi, identification, and their characterization depending on their functional role are discussed and provided as a helpful toolbox for a new researcher in the endophyte field.

Keywords

Endophytic fungi; Endophytic bacteria; Isolation; Identification; Characterization

1.1 Introduction

The plant microecosystem primarily consists of bacteria and fungi (Fisher et al., 1992). Among them, endophytic bacteria/fungi reside asymptomatically in plants and develop a mutualistic relationship with them. However, endophytes can sometimes change their lifestyle and turn into a pathogen, which is harmful for the host (Martinez-Klimova et al., 2017). The term endophyte has originated from Greek words “endon,” that is, within and “phyton” which means plant (Wilson, 1995). Endophytes are omnipresent, and are isolated from a wide variety of plants including bryophytes, pteridophytes, gymnosperms, and angiosperms (Bragina et al., 2012; Zhikai et al., 2017; Proença et al., 2018; Sun et al., 2008).

Most of the studies have described a symbiotic relationship between the plants and endophytes, however, the existence of some latent pathogenic endophytes cannot be disregarded (Bacon et al., 2008). Mainly, endophytic bacteria/fungi benefit from the high nutrient availability inside the plant while the plants gain through biotic and abiotic stress tolerance, growth promotion, and nutrient accessibility (Martinez-Klimova et al., 2017). Endophytes can provide protection to the plants from pests, herbivores, and even harmful microorganisms by utilizing the quorum-sensing system and prevent the pathogens from colonizing or by producing secondary metabolites (Kusari et al., 2015; Hartley and Gange, 2009; Hardoim et al., 2015). They can also increase the levels of plant growth-promoting (PGP) hormones (auxin, cytokines, and gibberellins) and facilitate the uptake of some nutrients such as nitrogen and phosphorus by solubilizing them from a complex form (Santoyo et al., 2016; Lambert and Joos, 1989). A plethora of endophytic-produced substances have been proved to be valuable for uses in medicine, agriculture, and lately in bioremediation, exploiting the heavy metal-resistant endophytes (Martinez-Klimova et al., 2017; Lin and Xu, 2013; Golinska et al., 2015).

Most of the endophytes specifically those that cannot survive outside of the plants are quite difficult to grow in laboratory settings. Thus, isolation and cultivation processes differ among the endophytic species and the right choice of the method can greatly affect the result. Advances in molecular techniques have simplified the isolation and identification of some unculturable species (Thomas et al., 2008; Podolich et al., 2009). The approaches that have been used in order to isolate and characterize endophytic organisms may seem quite diverse but with a more careful view they all follow the same rationale. In this chapter, we have made an attempt to summarize the commonly used methods and techniques for the sample preparation and isolation of endophytes to the identification of the specific species followed by their molecular and biochemical characterization.

1.2 Sample collection and preparation

Endophytes are isolated from various plants ranging from mosses to woody trees such as citrus (Zhikai et al., 2017; Gardner et al., 1982; Strobel et al., 2004). All the organs of the plant are possible niches for the endophytic microorganisms, while the roots possess the highest density of endophytes (Lodewyckx et al., 2002). Another factor that needs to be considered for plant selection is the health of the plant. Collecting samples from a healthy plant without visible symptoms reduces the chance of isolating pathogens together with the endophytes (Kusari et al., 2015).

The samples should be processed within 24 h of collection from the desired plant. However, the plant material can be stored at 4°C in sealed plastic bags to keep the tissue fresh and avoid death of the microorganisms (Gardner et al., 1982). After the collection of the samples, the surface sterilization procedure is essential in order to ensure that the isolated microorganisms are endophytes and not epiphytes (Verma et al., 2009). The schematic representation of the procedure for surface sterilization of plant material is shown in Fig. 1.1. Briefly, the surface sterilization starts with a repeat rinse of 5–10 times with sterile water followed by a wash of the plant tissue in 70%–75% ethanol for several minutes and finally rinse again with distilled water under a laminar hood. Sometimes additional steps of washing with sodium hypochloride (NaOCl) or sodium thiosulfate (Na₂S₂O₃) could be added for a more thorough sterilization (Qin et al., 2009). After the final wash of the plant material, some amount of the used water can be used as a negative control; a successful sterilization is indicated by the absence of microbial growth on the agar plate inoculated with this water sample (Shi et al., 2013). The sterile plant materials are cut into small pieces and then transferred to the growth media for the isolation of the desired microorganism.

Fig. 1.1 Procedure for sample preparation and endophyte isolation. The desired plant sample is being collected, thoroughly sterilized with water and ethanol and then fragmentized into small pieces. Fragments of the plant tissue are plated on growth media suitable for bacteria/fungi and incubated until colonies appear. Sub-culture the colonies in new plates until pure isolates acquired.

1.2.1 Isolation and identification of endophytic microorganisms

A great diversity of endophytic bacteria/fungi have been isolated from various plant tissues. Almost 70% of the endophytes that have been currently isolated belong to the fungi kingdom and the rest 30% are bacteria, based on the sequences deposited in the NCBI nucleotide database (Fig. 1.2). The common rationale involved in the isolation of endophytic bacteria/fungi is shown in Fig. 1.1. The preliminary steps, from collection of samples to surface sterilization and fragmentation of plant tissue, as described in above section are the same for both bacteria and fungi. However, the later procedures like the selection of growth media, antibiotics, incubation period, and molecular marker are different for bacteria and fungi, the details are described in the following sections.

Fig. 1.2 Proportion between isolated cultured/uncultured fungi and bacteria (as of February 4, 2019^α). In all, 70% of the isolates are fungi of which 81.3% are culturable and 18.7% are unculturable fungi (57% and 13%, respectively, in t...