STUDIES ON SOME OF THE ACTIVITIES OF MYCOFLORA ON THE RHIZOSPHERE AND RHIZOPLANE OF GLYCINE MAX (L.) MERRIL (SOYA BEAN).

ABSTRACT

Glycine max (L.) Merril (Soya bean) is one of the most important crops worldwide.   It is a versatile grain legume because it has a variety of uses.  A nutrient – dense food rich in protein, vegetable oil and essential minerals.  The crop has the ability to fix atmospheric nitrogen and thereby improving soil fertility.  Fungal mycoflora associated with the rhizosphere, rhizoplane, and non-rhizosphere of ten varieties of soya beans were isolated and studied. Dilution plate method was used in the isolation and a total of eighteen fungi were isolated at different developmental stages of the varieties. The morphological and molecular characterization of the isolates were carried out. The percentage frequencies of occurrence of the isolated organisms were determined. The organisms were screened for antagonism using dual culture technique. The interaction of variety, developmental stages and soil locations on the mycoflora were also determined. The isolated fungi were Aspergillus niger Van Tieghem,  Aspergillus fumigatus Fresenius, Aspergillus flavus Link ex Grey, Aspergillus tamarii Kita , Aspergillus hortai (Langeron) Dodge, Aspergillus nidulans (Eidam) Wint, Aspergillus arcoverdensis Sigler, Rhizopus delemar Fischer, Penicillium pinophilum Link. Fries, Paecilomyces lilacinus (Thom) Samson, Blastobotrys proliferans Fulvescens (Cooke) Apinis, Talaromyces pinophilus (Hedgcock) Samson, Neosartorya fischeri (Welmer) Malloch and Cain, Botrytis cinerea Pers., Mucor micheli ex Staint-Amans, Fusarium oxysporum Link ex Fries, Helminthosporium solani Link ex Fr., and Trichoderma asperellum Persoon ex Grey. The most predominant genus in all the varieties and soil locations was Aspergillus. Fusarium oxysporum, Mucor micheli, Neosartorya fischeri and Rhizopus delemar were also strongly present in all the varieties. Aspergillus fumigatus was the least in occurrence and it occurred only on rhizosphere and non- rhizosphere soils of V2 (two unrolled trifoliate leaves) developmental stage of varieties TGM 1055 and TGM 861 respectively. Maximum frequency of occurrence of fungi was obtained from the  rhizosphere  soil  of  soya  bean  varieties.  Fungal  colonies  at  vegetative  stages  were significantly different (P<0.05) and higher than the colonies at the reproductive stages. The interactive effects of variety, developmental stages and soil locations on the colony forming unit of the mycoflora were more significant (P<0.05) at vegetative developmental stages than at reproductive stages. The interactive effect of TGM 987 with RS (Rhizosphere) and V4 (four unrolled trifoliate leaves) developmental stage gave the highest colony of 49.7±11.26 followed by  TGM  861  which  interacted  with  RS  and  V6  developmental  stage  to  give  a  colony  of 29.67±15.06 for H. solani. The potential antagonism of T. asperellum against all the isolates except R. delemar was evidenced by the results. The inhibitory effects of T. asperellum on the isolates ranged from 7.83 % to 83.88 %. T. asperellum was most virulent against B. cinerea with the maximum percentage inhibition of 83.88 % while the least inhibited was R. delemar (7.83 %). The results showed that soya bean varieties harbour diverse fungal communities which could have positive influence on the growth of the plant and the bio-control agent (T. asperellum) which can be exploited for antagonism against plant pathogenic fungi.

CHAPTER ONE

INTRODUCTION

1.1Rhizosphere and Rhizoplane

The rhizosphere is a microecological zone in direct proximity of plant roots. It represents a highly dynamic front for interactions between roots and microflora. The rhizosphere of plant is functionally defined as the soil that adheres to roots (Plate 1A) after being gently shaken in water (Bonkowski et al., 2009; Bharti and Pravesh, 2012). The actual extent of the rhizosphere is dependent on the zone of influence of the plant roots and associated microorganisms. The rhizosphere is a metabolically busier, faster moving and more competitive environment than the surrounding soil. This area of soil is considered to be the most biodiverse and dynamic habitat on earth (Hinsinger et al., 2009).

The root surface and its adhering soil are termed the rhizoplane (Eze and Amadi, 2014). Sylvia et al. (2005) and Singer and Donald (2006) defined rhizoplane as the root epidermis and outer cortex where soil particles, bacteria and fungal hyphae adhere (Plate 1B). It is practically defined as the remaining microorganisms and soil particles after the roots have been shaken vigorously in water. There are more microbes in the rhizosphere than in the bulk soil. This is determined by counting the number of colony forming units (cfu) which are determined by spreading extracted soil microorganisms across an agar and counting the number of independent clusters of microorganisms (Sylvia et al., 2005).

Microorganisms are ubiquitous in nature and play a very important role in growth and establishment of plant in its natural environment. It can form parasitic or symbiotic relationship with its host plant. Fungal proliferation in the rhizosphere and rhizoplane occurs in response to the input of organic compounds exuded by the roots (Bharti and Pravesh, 2012). However, soil factors such as moisture, influence the amount of exudation and hence the colonization of the roots. In plants, exudates can have a healing and defensive response to repel insect attack or it can have an offensive habit to repel other incompatible or competitive plants (Sylvia et al., 2005).

1.2 Physical Environment

Plant roots have strong effects on the physical environment in the rhizosphere. Shifts in the physical environment, which are influenced by root manipulation, have significant impacts on the availability and form of substrates used for microbial metabolism. It also affects the prevalence of different microbial functions, as well as their total biomass within the rhizosphere. This goes both ways, as manipulation by microbes plays a strong role in the ways that plant roots interact with, benefit from, and influence the soil environment around them (Nihorimbere et al., 2011).

1.2.1 Water Potential

The  availability  of  water  is  a  major  component  in  determining  the  diversity  and prosperity of soil organisms. This is quantified by the water potential that explains the transport and energy potential state of water in different forms and locations (Philippot et al., 2013). As water is transpired across the leaf surface, tension is created within the leaf that is ultimately transferred  down to  the root  system.  This can  alter the water potential  of the rhizosphere, resulting in large fluctuations in the availability of water and solutes for microbes (Philippot et al., 2013). This fluctuation in available water can put pressure on the diversity and total biomass of microbes in the rhizosphere, by selecting for those with better mechanisms for water uptake (Sylvia et al., 2005).

On the average, 50 % of soils consist of pore spaces, composed of air and water with differing concentrations of solutes (Scow, 2015). The constituents of the soil solution provide the substrates necessary for microbes to thrive, ultimately determining the types of biotic and abiotic interactions taking place. Within the rhizosphere, solute activity is significantly higher than that of the bulk soil. The reason is that plant roots secrete ions and organic compounds that interact with organisms and other molecules in the environment around them (Philippot et al., 2013).

1.2.2 Soil Texture

The relative ratio of sand, silt and clay has large impacts on the availability of water, air and nutrients for microbial functions. The movement of roots through the soil profile occurs more rapidly in sandy soils than in clayey soils. This is due to sand having larger pore spaces between granules, allowing nutrient and water percolation to occur more rapidly. Increased porosity also allows for plant secreted compounds to extend further away from the root membrane. Therefore, the larger the granule size, the further the associated rhizosphere microorganisms will extend into the surrounding soil (Kent and Triptett, 2002). This means that larger granules require microbes to travel further distances to obtain nutrients or engage in interactions with plant roots (Sylvia et al., 2005).

1.2.3    Soil pH

The effects of pH extend to almost every characteristics of soil that are affected by multiple interactions within the rhizosphere. An example is respiration, a reaction that leads to carbon dioxide generation (Scow, 2015). In addition to the respiration of the roots themselves, the carbon rich environment of the rhizosphere promotes high levels of respiration by other macro -and microorganisms to a far greater extent than what is occurring in the bulk soil. The influencing effects of pH in the rhizosphere are critical in supporting a biologically diverse microbial community. Manipulation of the pH by plant roots is constantly occurring and while it is commonly believed that plants manipulate the soil pH in order to change the availability of nutrients. It also undoubtedly influences the abundance and diversity of associated microorganisms (Kent and Triptett, 2002).

1.3 Plant – Derived Compounds

Plant  –  derived  compounds  are  responsible  for  providing  the  additional  carbon  that allows the rhizosphere to host such a large variety of microorganisms. Plants have mechanisms to control the production and deposition of these compounds into the rhizosphere. These plant- derived compounds fall into five predominant categories: exudates, secretions, mucilages, mucigel, and lysates (Sylvia et al., 2005).

Exudates – These include surplus sugars, amino acids and aromatics that diffuse out of root cortex cells into the intercellular spaces and surrounding soil. They comprise the largest category of root secretions and are therefore considered to have the broadest effect in rhizosphere manipulation  (Jones  et  al.,  2009).  The  root  controls  the  rate  of  exudate  release  into  the rhizosphere through production of solute-specific membrane channels. The best known examples of exudates are organic acids that increase Phosphorous (P) solubility and reduce Aluminum (Al) toxicity (Jones et al., 2009). Due to their diffusive nature, exudates are limited to compounds of low molecular weights (Bakker et al., 2013).

Secretions – These are also known as secondary metabolites. They are by-products of metabolic activity. Because they are released from the cell via active transport, secretions can be of both low and high molecular weight. They are also important in increasing mobilization of insoluble compounds such as Iron (Fe) and Phosphorous (P) (Jones et al., 2009).

Lysates – They are lysed contents of a cell which are released into the surrounding soil. When an epidermal root cell dies and is broken open, lysates from within the cell become available to the surrounding microbial community. These components include proteins, nucleic acids, lipids and various forms of carbohydrates (Hartmann et al., 2009; Lakshmanan, 2014). Lysate compounds are important in maintaining the C/N ratio of soil organic matter (Jones et al., 2009).

Mucilages – These are the results from abrasive forces of the root against soil particulate matter that removes cells from the root cap. These newly removed cells consist of cellulose, protein, starch, and lignin, in which they are often highly recalcitrant and therefore contribute to higher diversity of carbon decomposers (Lakshmanan, 2014)

Mucigel– This is a slime coating the surface of a root that increases the connectivity between plant roots and the surrounding soil. It is more common on the main body of the root and root hairs than on the tip. During very low soil water potential, mucigel is responsible for allowing plants to continue the uptake of water and nutrients, as well as reducing the negative effects of desiccation (Sylvia et al., 2005).

1.4 The origin of Glycine max (L.) Merril

Glycine max, commonly known as soybean in North America or Soya bean in British English is a species of legume native to East Asia (Anonymous, 2008).  It was first domesticated in Northeastern China around the 11 century BC.  The crop spread to Manchuria, Korea, Japan, Russia and to other Asian countries.   It was introduced into United States of America in the beginning of 12th  century as hay but later was cultivated for oil seeds and is now the most important  cash  crop  of  the  country  (Masuda  and  Goldsmith,  2009).  Soya  bean  was  first introduced to Ibadan, Oyo State, Nigeria in 1908 with little or no success in the rainforest ecology of the State (AMREC, 2007).  In 1928, it was introduced to the savanna area of Northern Nigeria where the soil and climatic conditions supported its production.  Presently Nigeria is the largest producer of the crop for human and livestock feeds (AMREC, 2007). The major soya bean producing States in the country are Benue, Kaduna, Taraba, Plateau and Niger.   Other growing areas include, Nasarawa, Kebbi, Kwara, Oyo, Jigawa, Borno, Bauchi, Lagos, Sokoto, Zamfara and FCT (AMREC, 2007).

1.5 Taxonomy of Glycine max (L) Merril

Soya bean is scientifically classified as follows: Kingdom                        –           Plantae

Phylum           –          Spermatophyta Sub-phylum  –        Magnoliophyta Class               –          Magnoliopsida Order              –          Fabales

Family            –          Fabaceae Subfamily        –         Faboideae Genus             –          Glycine Species           –           max Source: USDA (2016)

1.6 The Botany of Glycine max (L.) Merril.

Glycine max, the soya bean (also known as soya or soja bean, formerly classified as Glycine  soja),  is  an  annual  herbaceous  plant  in  the  Fabaceae  (legume  or  bean  family).  It originated from Southeastern Asia (including China, Japan, and Korea) and was domesticated more than 3,000 years ago for its edible seeds and young pods (Wyk, 2005). It is now the world’s most important legume crop, and the sixth of all cultivated crops in terms of total harvest, and the most widely produced oilseed, grown in diverse climates worldwide (Henkel, 2000).

The soya bean plant, which is densely hairy on leaves and stem, can grow to nearly 1.82 m tall, leaves are compound, with 3 leaflets. The inconspicuous, stalkless white to purple flowers are borne singly or in small clusters in the leaf axils. The fruit is a broad, hairy, flattened legume or pod, about 10 cm long, yellow to brown when fully mature and dried. Pods typically contain up to 4 beans, which vary in size and colour depending on cultivar (colours range from white to reddish to black) (Shurtleff and Aoyagi, 2013).

1.7       Importance of soya bean in the global economy

Soya bean is among the major industrial food crops that can be successfully grown in every  continent.  The  crop  can  be successfully  grown  in  many states  in  Nigeria using  low agricultural input (Dugje et al., 2009). Soya bean cultivation in Nigeria has expanded as a result of its nutritive and economic importance and diverse domestic usage. It is also a prime source of vegetable oil in the international market. Soya beans are considered by many agencies to be a source of complete protein (Henkel, 2000). A complete protein is one that contains significant amounts of all the essential amino acids that must be provided to the human body because of the body’s inability to synthesize them. For this reason, soya bean is a good source of protein amongst many others, for vegetarians and vegans or for people who want to reduce the amount of meat they eat (Wolf, 2012). Soya bean has an average protein content of 40 % and is more protein rich than any of the common vegetable or animal food sources found in Nigeria. Soya bean seeds also contain about 20% oil on a dry mater basis, and this is 85% unsaturated and cholesterol-free (Dugje et al., 2009).

1.8 Problem Statement

•   The  rhizosphere  is  a  complex  ecosystem  made  up  of  numerous  communities  of microorganisms. Its diversity and benefits are yet to be fully understood.

•   Micro organisms growing on the plant roots can influence plant growth positively or negatively therefore the study of differences in microbial colonization among plant varieties may have practical implications. Such practical implications may include effect on the productivity of the plant.

•   The effects of continuous agricultural practices such as fertilization can cause serious damage to the environment.

•   Some fungi cause a range of plant diseases, while others antagonize plant pathogens, decompose plant residues, provide nutrients to plants, and stimulate plant growth.

1.9       Justification of the study

Some microscopic fungi have shown considerable promise for root pathogen control, which calls for knowledge of the regularities of their colonization, development and distribution in the rhizosphere and rhizoplane.

Therefore, it is believed that soya bean production will increase as more farmers become aware of the potentials of the crop, not only for cash or food but also for soil fertility improvement.

1.10 Objectives.

The objectives of the study were:

•   To isolate, identify and characterize to molecular level different fungal populations that colonize the rhizosphere, non-rhizoshere and rhizoplane of soya bean at different growth stages.

•  To determine physicochemical properties of the soil at the selected growth stages.

•   To find out effects of variety, developmental stages and locations on the colony of the isolates.

•  To determine antagonistic pathogens.

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