Abstract

Endophytic fungi inhabit healthy plant tissues, and are now considered as ubiquitous symbionts of plants. Besides having the positive effects on the plants in phytostimulation, production of pigments, enzymes and bioactive compounds and nutrient cycling, they are also responsible for the destruction of host tissues and toxic production within the host. The endophytic fungi can be found in any part of the plant such as scale primordial, meristem, resin ducts, petiole, buds, stem, root, shoot, leaves, barks and even in the pneumatophores. Mangroves act as a host for plenty of endophytic fungal populations. The endophytic fungi obtained from mangroves have certain specialized characteristics and are resistant to several environmental stress conditions. Aim of the present study is to isolate and identify endophytic fungi inhabiting mangrove plant species that are abundantly found along the state of Northern Kerala, India. The collected plant specimens were surface sterilized and ground to paste form to earn the extract, which was then inoculated into the medium to obtain fungal culture. Among seven plant specimens collected, six types of endophytic fungi were morphologically identified as Aspergillus sp. (two different species), Penicillium sp., Alternaria sp., Fusarium sp. and Sarocladium sp.

1. Introduction

Mangroves are coastal rainforests regarded for their adaptability to harsh environmental conditions such as high salinity, temperature fluctuation, strong winds, extreme tides and even low oxygen levels. They are also known as tidal forests or coastal woodlands. Mangroves are tropical and subtropical forest systems which include flora from different genera sharing the common characteristics to grow in marine ecosystem including estuarine ¹. The word mangrove is a combination of Portuguese word “Mangue” and English word “Grove” ². In India, mangroves cover an area of 4,639 sq. km, which is 0.14% of the total geography of the country. However, the mangrove cover in India represents 3% of the global mangrove cover and 8% of Asian mangrove coverage ^{3, 4}. East coast along with Bay of Bengal occupies about 60% of the Indian mangroves, West coast in Arabian Sea possesses around 27% and Andaman and Nicobar Islands show13% of mangroves. In India, the maximum mangrove cover is in Sundarbans, followed by Gujarat and Andaman and Nicobar Islands ⁵.

The state of Kerala provides favorable environment for the rich growth of mangroves with its coastal line of around 560 km and 41 rivers, emptying into the Lakshadweep Sea. Out of all 14 districts of Kerala, 10 districts have mangrove covers in the coastal environment. Districts like Idukki, Pathanamthitta, Palakkad and Wayanad are places having no coastal areas and hence do not have mangrove forests. Among the districts having mangrove cover, Kannur has the richest floristic diversity of mangroves ⁶. The district has the distribution of about 83% of mangrove stocks of Kerala. The mangrove species that are abundantly found in Kerala are Rhizophora mucronata, Rhizophora apiculata, Avicennia officinalis, Avicennia marina, Bruguiera cylindrica, Kandelia candal, Sonneratia alba, Sonneratia caseolaris, Excoecaria agallocha and Aegicera scorniculatum ^{7, 8}.

Fungi could be either endophytes or ectophytes. Ectophytes are those that reside outside the host body, whereas endophytes are organisms that grow inside the body of the host organism. Endophytic fungi are a group of highly diverse fungi falling in the class ascomycetes. They are found within the plant tissues without causing any notable effects ^{9, 10, 11}. Nearly 3,00,000 species of higher plants exist on earth and each plant contains diversity of endophytes ¹¹. In most cases, fungi induce some diseases on the host organisms or produce reproductive structures at host’s expense. But in case of endophytic fungi, they do not show any external signs of growth on the plants. These endophytic fungi could be isolated and identified by microscopic observation or molecular analysis according to laboratory protocols. Endophytes prevent the growth and development of pathogenic organisms within the plant tissues. They are classified into two groups as clavicipitaceous and non-clavicipitaceous based on the differences in their selection of the host plant, ecology, evolution and taxonomy. Clavicipitaceous are those endophytes that can infect only grasses whereas non clavicipitaceous are endophytes found in the asymptomatic tissues of non-vascular and vascular plants. Endophytes enhance plant growth, distribute soil nutrients and increase resistance to abiotic stress in plants ¹². Aspergillus sp. was found to be the dominant endophytes among the various fungal species isolated from the mangrove ecosystems ¹³.

The other commonly found endophytes isolated from mangroves are Fusarium sp. and Penicillium sp. Endophytes may produce abundant potential substances that can be used in agriculture, industry and modern medicine such as novel antibacterial, antifungal and anticancer compounds and immunosuppressants ^{14, 15}. Some of the factors that influence the endophytic fungal communities include geographical area, climatic conditions, host physiology and specificity of the colonized plant tissue ^{5, 16}. Endophytic fungi commonly protect their hosts against pathogens, insects and pests ¹⁷. Seena and Sridhar ¹⁸ worked on two sand legumes from south coast of India to study the endophytic fungal diversity. They concluded in their study that there is no significant difference in the endophytic fungi colonization between plant species. Suryanarayanan et al. ¹⁹ isolated endophytic fungal species from the leaves of Rhizophora apiculate Bl. and Rhizophora mucronata Lamk., collected from Pichavaram mangrove at Tamil Nadu. They compared the richness index of isolated endophytic fungal diversity between two plant species collected in two different time periods. They observed that the presence of the endophytic fungal diversity is higher in rainy days than in summer days.

2. Materials and Methods

The samples were collected from two mangrove sites at Valiyaparamba of Kasargod district and Kunhimangalam and Ezhimala of Kannur district. Healthy and mature plant species were chosen for sampling. The bark samples were obtained aseptically and were stored in sterile polythene bags ¹⁹.

The plant materials were rinsed with tap water to remove dust and debris and then washed with sterilized water to cut into small pieces aseptically. The cut pieces were surface sterilized with 70% ethanol for three minutes and washed again five times using sterile distilled water. These surface sterilized samples were weighed. About 0.5g of each sample were ground using a sterile mortar and pestle by adding 2ml of sterile distilled water and a paste of the sample was obtained. About 0.1 ml paste of the sample was then plated onto Potato Dextrose Agar (PDA) and was incubated at room temperature for 4 days ^{12, 18}.

The fungal colonies were stained using lactophenol cotton blue dye. The prepared slides were viewed under trinocular light microscope. The fungal species were identified on the basis of their morphological characteristics. The different fungal isolates were described on the basis of their macroscopic (topography, texture and colour) as well as microscopic (structure of hyphae, conidia, conidiophores and phialides) characteristics ^{20, 21}.

3. Results and Discussions

The study areas selected were Valiyaparamba situated at 12.1510°N latitude and 75.1420°E longitude in the belt of Kasaragod district, Kunhimangalam situated at 12.0718°N latitude and 75.2285°E longitude and Ezhimala situated at 12.0213°N latitude and 75.2071°E longitude in the belt of Kannur district. Table 1-2 shows the seven mangrove plants species that were obtained from the sampling sites and its characteristics respectively. The collected plant species were identified based on literature ^{2, 5} as Avicennia officinalis, Bruguiera cylindrica, Excoecaria agallocha, Acanthus ilicifolius, Kandelia candel, Rhizophora apiculata and Aegicera scorniculatum (Figure 1).

Each species of plants harbor a specific kind of endophytic fungi. In the current study, fungal species were isolated from barks of the specimens. Plant parts of seven broad ranges of mangrove plant species were collected and processed for the isolation of endophytic fungi. Six different fungal species were observed under the trinocular microscope. The characteristics shown by each fungal species microscopically and macroscopically were noted and identification was done on the basis of their observed characteristics (https://mycology.adelaide.edu.au) ²². Macroscopic and microscopic views of the isolated fungal species are given in Figure 2 and Figure 3 respectively. Table 3 explains the macroscopic and microscopic characteristics of the isolated fungal species. A total of six fungal strains were obtained from the samples of which 3 belong to the class Eurotiomycetes and each one out of the remaining three are from the classes, Dothideomycetes, Sordariomycetes, and Ascomycetes. The isolated endophytic fungi were confirmed with their microscopic and macroscopic features as Penicillium sp., Alternaria sp., Fusarium sp., Sarocladium sp. and two Aspergillus sp. (Table 4). Table 5 shows the identified fungal endophytes and the respective plants from where they have been isolated.

From the surface sterilized bark samples collected from seven mangrove species, six endophytic fungi were identified. Among the six endophytic fungi, Fusarium sp. was found to be the most dominant fungi due to its presence in all seven plant species obtained. Second most dominant species was Penicillium which was isolated from all collected plant species except Excoecaria agallocha. Aspergillus sp. was obtained from Bruguiera cylindrica and Rhizophora apiculata. The genus Alternaria was obtained from Aegiceras corniculatum, Rhizophora mucronata and Excoecaria agallocha. Sarocladium sp. was found only in Aegiceras corniculatum and Excoecaria agallocha. Another different species of Aspergillus from previously mentioned was isolated from only Acanthus ilicifolius.

Though the endophytic population is highly diverse, it shows uniqueness depending upon the host plant it occupies. Mangroves being peculiar kind of plants growing in saline and moist environment, houses a wide variety of endophytic fungal population. Endophytes act as mutualists ²³ and are present in almost all parts of the plant. The fungal diversity present in each plant part also varies. This study identifies the endophytic fungal population present in the bark samples of the mangrove plants abundantly seen along Northern Kerala.

4. Conclusion

The endophytic fungi create their own habitat and ecological niche inside the plant cells. They reside inside the plant cells without causing any effect on the plants. These fungi can interact positively and negatively with their environment. They have great potential in the drug and pharmaceutical industry as most of the fungal strains possess antimicrobial, antibacterial and anticancerous properties. Studies regarding endophytes have proved that they can decrease insect herbivory, enhance plant growth, increase drought resistance and disease resistance. Endophytic fungi are also recognized as a major source of novel bioactive compounds and secondary metabolites useful in biological control ²⁴. With regard to all these, endophytes prove that they have beneficial impacts on plants, environment and humans in different ways. They can be used to fight against harmful pathogenic organisms in the agricultural, pharmacological and drug development fields. In conclusion, the present study was conducted during summer season which may be the reason for less endophytic fungal species isolation. It is supported by Suryanarayanan et al. ¹⁹. They stated that diversity richness of endophytic fungi would be higher during rainy months than summer. The present study will be strengthened by carrying out the analysis throughout the year in different seasons with molecular and phylogenitic analysis in near future.

Statement of Competing Interest

The authors have no competing interests.

References