Isolation, Identification and Characterization of Fungus causing Coconut’s Bud Rot Disease: Inhibition using Silkworm Fecal Matter Mediated Synthesized Nanoparticles

Introduction

Nanomaterials are cornerstones of Nano-science and nanotechnology. We specifically considered nanoparticles as clusters of atoms in the size of 1-100nm. ‘Nano’ is a Greek word synonymous to dwarf meaning extremely small. Nanostructure science and technology is expansive and interdisciplinary area of research and development activity that has been growing explosively worldwide in the past few years. The idea of nanotechnology was coined by physicist Professor Richard Feynman (known as “Father of Nanotechnology”) in his historic talk “there’s plenty of room at the bottom” Feynman RP, et al. [1], though the term nanotechnology was introduced by Tokyo Science University Professor Norio Taniguchi Taniguchi N, et al. [2]. Nano-biotechnology has emerged as integration among biotechnology and nanotechnology for developing biological synthesis and environmental-benign technology for synthesis of nanomaterials. Nano-biotechnology is one of the most promising areas in modern Nano-science and technology. Biological synthesis is a green and environmental friendly approach of synthesizing nanoparticles which are biodegradable and non-toxic to the environment [3]. Biosynthesis includes using of plants, algae, fungi, actinomycetes, yeast, bacteria, etc. along with precursors to synthesize nanoparticles alternative to convention chemicals for capping and bio reduction purposes. The biologically synthesized nanoparticle finds their own way in biomedical applications due to their unique and enhanced properties [4]. The wide variety of core materials available, coupled with tunable surface properties, make nanoparticles an outstanding platform for a broad range of biomedical and biological applications.

The  coconut tree  (Cocos nucifera) is a member of the  palm tree  family  (Arecaceae) and the solitary living species of the genus Cocos. The term “coconut” (or the archaic “coconut”) can refer to the whole coconut palm, the seed or the fruit, which botanically is a drupe, not a nut. The term is derived from the 16th-century Portuguese and Spanish word coco meaning «head» or «skull» after the three indentations on the coconut shell that resembles facial features. Coconut belongs to Kingdom: Plantae, Order: Arecales, Family: Arecaceae, Subfamily: Arecoideae, Genus: Cocos, Species: nucifera. In India it is cultivated in an area about 1.93million hectare with an estimated production of 12,147million nuts, has tremendous influence on the socio-economic property of million people. Coconut palm is dominant and essential component of the homesteads and garden lands along the coastal parts of southern India and it plays a vital role in the sociocultural and economic life of huge number of small and marginal farmers Dagar JC, et al. [5]. Coconut tree has much economic importance as food, drink, health, shelter, medicine, fuel, aesthetics and wealth. Because of its versatile nature, it is known as “Kalpa-Vriksha”, “Tree of abundance”, “Tree of Heaven”, “Tree of Life”, “Nature’s super market”, “King of palm”, “Tree of virtues “ and by many names. It is also known as lazyman’s crop Gupta S, et al. [6]; Loomba S, et al. [7]; Lima EBC, et al. [8].

Coconut production is limited by several factors, among them diseases play a major havoc role. Coconut crop diseases are a major biotic stress, play a great havoc causing considerable reduction in yield level. Due to microbial infections, there were several major diseases reported from our country, causing severe damage to the plant body such as Basal stem rot (Thanjore wilt), Bud rot, Leaf blight, Leaf rot disease, Root wilt, Stem bleeding Nambiar KKN, et al. [9]; Louis H, et al. [10]. Among those Bud rot disease caused by Phytophthora palmivora was known to be severe of coconut palm. Palms of all ages were susceptible to bud rot disease, but the young palms were observed to be more susceptible, particularly during monsoon season when the temperature is low and humidity is very high. Briton-Jones HR, et al. [11] described the disease symptoms first. The first visible symptom was the withering of the spindle marked by pale colour. The spear leaf or spindle turns brown and bends over. A basal tissue of leaf rots quickly and can be easily detached from the crown. Spindle withers and droops down and one by one, the inner leaves also fall away, leaving only fully matured leaves in the crown. A foul smell is emitted by the rotting tissue [9]. The palms succumb to the disease with the death of the spindle Briton-Jones HR, et al. [11]; Menon KPV, et al. [12]; Lingaraj DS, et al. [13]. Later, infection spreads to the older leaves, causing sunken leaf spots covering the entire leaf blade spreading both up and down. Spot margins are irregular and water soaked and when the leaves are unfolded, the characteristic irregular spots are conspicuous on the blade. In severely affected trees, the entire crown may rot and in few months the trees wilt. The heart leaf becomes wilts, chlorotic and collapses. The disease may spread to older, adjacent leaves and spathes, producing a dead center with a fringe of living leaves. Light brown to yellow, oily, sunken lesions may be seen on leaf bases, stipules or pinnae. Internally, the tissues beneath the bud rot discolored pink to purple with a dark brown border. Infected nuts showed brown to black necrotic areas with yellow border rising on the surface. Internally, they have a mottled appearance. Young nuts are highly susceptible to disease and fail to mature and then fall off from the tree. Older, infected nuts ripen normally [14, 15]. Ultimately, the entire crown falls down and the palm dies. In the 1920s “bud rots diseases” were identified in Jamaica, Puerto Rico, Africa, Peninsular Malaysia and the Philippines [12]. Later, it was also reported in India, Sri Lanka, Central America, West Indies, Fiji, and Vanuatu etc. Several practices like spraying chemicals, keeping the garden clean, application of talc powder formulation on rotten areas, etc., were made to avoid the spreading of bud rot disease [16].

Phytophthora is a genus of plant- damaging  oomycetes  (water molds), whose member species are capable of causing huge economic losses on crops worldwide, as well as  environmental damage  in natural ecosystems. Phytophthora palmivora is an oomycete which causes bud-rot of palms, fruit-rot orkole- roga of coconut and areca nut. These are among the most severe diseases caused by fungi and moulds in South India. The causative organism was first identified as Phytophthora palmivora by Butler in 1917. P. palmivora belongs to Phylum: Heterokontophyta, Class: Oomycota, Order: Peronosporales, Family: Peronosporaceae, Genus: Phytophtora, Species: palmivora. The pathogen was assumed to have originated in Southeast Asia but is now pantropical. It causes significant losses to farmers of tropical fruit and vegetable crops. P. palmivora infects a thousand or more plant species including horticultural, ornamental and agricultural crops. It is also a common soil inhabitant. Important horticultural hosts include cocoa (black pod, canker, cherelle wilt), papaya (fruit rot), durian (fruit rot, canker) pineapple (heart rot, citrus (canker), black pepper (foot rot) and coconut (bud rot). P. palmivora grow vigorously under humid wet conditions and as a result can cause significant losses in many economically important tropical fruit crops [16]. The main distinguishing feature of the pathogen includes conspicuous papillate sporangia, which can be distinguished from other Phytophthora species because they are caducous and have short pedicels. Primary inoculum originates from soil and infected plant material. The pathogen is circulated through rain splash, insects and human activity into the canopy of trees, where symptoms appear. Secondary inoculum spreads rapidly through wind and rainsplash, contact and vector activity in humid weather [17, 18]. Phytophthora palmivora can be managed using cultural and chemical methods. The use of resistant varieties, sanitation (including complete harvesting and the removal of infected planting material and weeds), and improved nursery hygiene, pruning to improve air flow and reduce humidity and boosting soil health by increasing organic matter can be used as part of an integrated management strategy. Chemicals used in the control of P. palmivora include metalaxyl, phosphonates or copper hydroxide to paint cankers, and phosphonates as a soil drench, trunk injection or foliar spray.

In present investigation, we have undertaken inhibition studies against isolated bud rot disease causing fungi using novel metal nanoparticles synthesized using Silkworm fecal matter, which would lead to develop a new drug and treatment for bud rot disease of coconut. This would in turn may helpful for uplifting the coconut growing farmers economically.

Materials and Methods

All the materials used in the present study were used as received. Chemicals used in the study were of analytical grade and highest possible purity. They were purchased from Hi-Media Chemicals (Mumbai, Maharashtra, India) and Sigma Chemical Company (St. Louis, Mo. USA).

Discussion

In the present investigation, the fungal species was isolated from bud rot affected coconut plant using PDA medium. The fungal species was identified as Phytophthora palmivora by its molecular characterization. The sequence of the isolated has shown upto 98% identity with other Phytophthora species. It is well-known that molecular classification is a fast procedure which requires least management of pathogens and also supports in distinguishing morphologically, similar fungal species. Similar applications of PCR technology were used for detection and identification of fungi, by using an). Phytophthora palmivora is the main cause for Bud rot disease in coconut, which causes huge loss to economy of farmers. Rashmi AR, et al. [21] and Sharadraj KM, et al. [22] and many other researchers reported the isolation of Phytophthora palmivora which caused bud rot and root rot in coconut. In 2008, Srinivasulu B, et al. [16] made a complete report on Phytophthora palmivora isolation from bud rot caused tissue of coconut and well written about the disease management [16]. Including Bud rot of coconut, P.palmivora is one of the reasons for many deadly diseases of other plants. In the same way, Hung PM, et al. [23] identified P. palmivora from root rot symptoms of Citrus maxima in Thailand. Serious root rot disease of citrus and papaya caused by P.palmivora has been recorded in India Graham JH, et al. [24] and America Zitko SE, et al. [25] also isolated and characterized Phytophthora species which caused Citrus Gummosis in Kenya. Suskiri S, et al. [26] reported Phytophthora form Durian Orchard in Chumphon Province, Thailand. Root rot and stem rot are the major disease of durian orchard, which is caused by Phytophthora species. Root rot of Pomelo caused by this pathogen was observed in Thailand [23]. There are many other major diseases are associated with Phytophthora species which causing major loss to agricultural and ornamental crops. Hence, the fungus should be inhibited to avoid loss of economy.

Biosynthesized nanoparticles showed an effective range of antifungal activity against Phytopthora palmivora. Antifungal activity of synthesized nanoparticles was determined using Radial Growth Technique [19]. As P.Palmivora shows strong dose dependent response over the addition of AgNPs and CuNPs. There was an increase in the percentage of inhibition with the increase in the concentration of biosynthesized nanoparticles. In present study, both AgNPs and CuNPs were proven to be good antifungal agents, where AgNPs established remarkable inhibition activity against the pathogenic fungi P.palmivora. When compare to CuNPs and standard Fungicide (Fluconazole). As previous reports suggests, biosynthesized nanoparticles were already proved that they were remarkable antifungal agents against many pathogenic fungus like Candida albicans and Aspergillus niger [27], Rhizoctonia solani, Fusarium oxysporum, Sclerotiniasclerotiorum and Sclerotium rolfsii Kaman P, et al. [28]; Khan NT, et al. [29] reported antifungal activity against Candida glabrata, Candida albicans and Candida tropicalis using fungal mediated nanoparticles in disc diffusion method [29]. However, there is a report available on antifungal activity of nanoparticles against pathogenic fungus causing red root rot disease in tea plants [30]. This study is the first trial of biosynthesized nanoparticles as antifungal agents against the bud rot causing pathogenic organism Phytophthora palmivora. Our inhibition study stands in front with the previous reports on inhibition of P. palmivora reported by Gills-Alex P, et al. [31].

Conclusion

Bud rot causing fungus was isolated from infected tissue of coconut using suitable method. The molecular characterization of the isolated fungus species as Phytophthora palmivora. Where in the sequence has 98% homology with the similar Phytophthora species. The isolated species was found to be the main reason for causing bud rot disease to coconut trees [32, 33]. Therefore, there was a demand for investigating to inhibit the Phytophthora palmivora for protecting the disease. In present research, the inhibition effect of AgNPs and CuNPs synthesized using silk worm fecal matter was explored against Phytophthora palmivora using radial growth technique. The pathogenic fungus showed dose dependent inhibition response on addition of nanoparticles. Here we have observed, both synthesized AgNPs and CuNPs were very excellent antifungal agents. The Percentage of Mycelial Growth Inhibition of fungus was 86%, 70% and 80% for AgNPs, CuNPs and Standard Fungicide respectively. For the concentration of 3mg/ml and AgNPs were found as potent antifungal agent against Phytophthora palmivora, when compare to CuNPs and standard Fungicide. Plant cell wall-degrading enzymes play significant roles throughout the fungal life including acquisition of nutrients and decomposition of plant cell walls. Many plant pathogens are known to secrete a variety of PCWDEs to perceive weak regions of plant epidermal cells and penetrate the plant primary cell wall. Cell wall degrading enzymes are believed to help pathogens directly penetrate  host  root  tissue. Therefore, may be essential for  pathogenicity. Cell wall degrading enzymes produced by pathogenic fungus were found to play an important role in penetration of organism inside the plant tissue; hence it is critical to inhibit those enzymes using synthesized nanoparticles. Our Silver and copper nanoparticles have successfully inhibited the cell wall degrading enzymes produced by pathogenic fungi in a dose dependent manner. Therefore these nanoparticles could be used as potent inhibitor of Phytophthora palmivora in place of standard fungicide fluconazole.