Root-Knot Nematode Management Using Chitin-Rich Fish Industry By-Product in Organic Brinjal Cultivation ^†

Abstract

Root-knot nematode, Meloidogyne incognita, is a silent enemy of brinjal and can cause fruit yield loss of up to 80% in organic brinjal cultivation. Effective management of M. incognita using synthetic nematicide could be detrimental to non-target organisms and the environment. The aim of this study was to explore the impact of chitin-rich shrimp and crab exoskeleton soil amendments on M. incognita growth. To this end, a green house experiment was conducted with and without shrimp and crab exoskeleton amendment. We recorded root-knot number, root and shoot length and fresh and dry weight after one month. The findings revealed that crab and shrimp chitin amendment-treated plants exhibited a significant reduction in the number of galls with the values of 15.5 and 25.75, respectively, than the control (35.5) at p < 0.05. The shoot length (14.90 cm), shoot fresh weight (5.39 g) and shoot dry weight (4.46 g) of brinjal were the highest in crab powder-treated plants at p < 0.05, whereas, root fresh (1.53 g) and dry weight (0.14 g) were highest in the control. We conclude that crab and shrimp exoskeleton powders suppress the M. incognita infestation and improve the growth parameters of the brinjal. Extensive field studies are needed to recommend chitin as an alternative to nematicides to manage M. incognita in organic brinjal cultivation.

1. Introduction

Solanaceae family member brinjal (Solanum melongena L.) is an economically important vegetable crop that provides diversified nutrients to low-income people in Asia and Africa [1]. Brinjal ranked among the top ten vegetables which have high antioxidants such as anthocyanins, phenolic acids, flavonoids, etc. [2,3]. Brinjal is cultivated more than 1800,000 ha around the world annually and the estimated annual yield is 50 million tons [2]. Soil-borne pathogens and pests are major biotic hurdles to the brinjal cultivation, especially in organic agriculture, and inflict 78% of the damage on brinjal production if not managed properly from the beginning of the cultivation [4].

Among the soil-borne pathogens, obligate endo-parasite root-knot nematode Meloidogyne spp. belongs to the family Tylenchid and is the most dangerous polyphagous pathogen attacking brinjal, causing an average loss of 30%. More than 70 species of root-knot nematodes have been identified around the globe, and among them, 95% of the infestations are only caused by four important species such as M. incognita, M. arenaria, M. hapla and M. javanica [5,6,7,8]. M. incognita’s existence is high in tropical regions, including the current research field, and causes economical loss to more than 2000 plant species [9,10].

Root-knot nematode M. incognita is present in soil and attacks the belowground part of the plant and is therefore responsible for major brinjal fruit yield loss. Management of nematodes is a difficult task due to its adaptation to vast environmental and soil conditions [10]. Chemical management of M. incognita is an effective method, but it leads to hazardous effects on the living environment such as humans, predators, pollinators and parasites such as beneficial organisms [11]. Hence, nematologists are investigating continuously to discover new avenues to manage the M. incognita in an eco-friendly manner [12].

The utilization of organic amendments is an alternative for the effective management of root-knot nematode. In particular, botanicals replace the synthetic nematicides. Mexican marigold leaf, bitter leaf, lantana leaf extracts and neem (Azadirachta indica) and thulasi (Ocimum sanctum) aqueous extracts inhibit the egg hatching [13,14]. Castor leaf and seed (Ricinus communis) and moringa leaf and seed (Moringa oleifera) extracts reduce the root gall index [15]. Syzygium aromaticum L., Nicotiana tobacum L. and Nerium oleander leaf extracts have the ability to directly immobilize the effect on second-stage juveniles of root-knot nematode [16,17].

The organic amendments derived from sewage wastage, livestock manure, poultry refuse, rice bran, tea waste and dry and fresh sawdust are very effective against root-knot nematode [18]. Composts produced from diversified farm wastes such as, goat manure, poultry manure and vermicompost proved to inhibit the nematode gall [18]. Refuge dump, rice husk and sawdust amended farm soil, exhibiting a 70–88% reduction in the root-knot nematode population [19].

Chitin is a polysaccharide with the polymer of unbranched chains of β-(1,4) linked 2-acetamido-2-deoxy-d-glucose residues. Chitin possesses nematicidal properties. Chitin amendments can be used instead of synthetic nematicides. It has been proven that chitin induces a conducive rhizosphere environment for beneficial microbes to act and produce copious amounts of secondary metabolite excretions which are hazardous to M. incognita [20,21]. Moreover, chitin and chitin-urea amendments release the ammoniacal nitrogen which acts as a nematicide and combines with the chitinolytic microorganism; therefore, it is good to use when practicing the fallowing period [22].

2. Materials and Methodology

2.1. Inoculum Maintenance and Extraction

Root-knot-nematode-infected plants and soil samples were collected from Thirunelvelly, Thanduvan and Omanthai by a random sampling method. The M. incognita pure population was maintained on Thirunelvelly-purple eggplant in the Department of Agricultural Biology, Faculty of Agriculture, University of Jaffna. Collected plant roots were washed under running tap water to remove soil and adhered particles. Galls on the roots were cut, peeled and subjected to microscopic examination. Pear-shaped female M. incognita was observed and carefully isolated by a fine camel hairbrush and scalpel.

2.2. Preparation of Natural Chitinous Amendment

The exoskeletons of shrimps and crabs were collected and the inner contents of the shrimps and crabs were removed. The cleaned exoskeleton was washed with tap water followed by distilled water. The exoskeletons were allowed to dry under sunlight for 3 days. Dried exoskeletons were ground and made into fine powder by a mortar and pestle separately.

2.3. Pot Preparation

The pot experiments were undertaken in seven kilograms of black polybags. Soil was sterilized and allowed to cool for two days before sowing. Five grams of each exoskeleton powder was mixed with potting media. It was kept to decompose for one week. Watering was carried out during the decomposition period.

2.4. Nematode Inoculation

After one week of decomposition of the amendments, three-week-old Thirunelvelly-purple brinjal seedlings were transplanted into the polybags. Two days later, seedlings were inoculated with fifteen matured pear-shaped female nematodes. The inoculation was carried out around the seedling root zone by making holes. The control pots were not given any chitin amendment treatments. Seedlings were allowed to reach growth performance for a month.

2.5. Observation and Data Collection

Daily mean temperature and rainfall were recorded. One month after the inoculation, the brinjal plants were uprooted carefully to avoid the damage and washed to remove adhere particles on root. Data on plant growth parameters such as fresh and dry weight of shoot and root (g), shoot height (cm) and root length (cm) as well as pathological parameters of root-knot index (RKI = 0–5 scale) were taken. Root-knot index (

Table 1. Effect of chitin amendments on Meloidogyne incognita.

Treatment	Length (cm)		Fresh Weight (g)		Dry Weight (g)
	Shoot	Root	Shoot	Root	Shoot	Root
Control	12.50 c	4.68 b	2.84 c	1.53 a	1.4 c	0.14 a
Shrimp	13.67 b	5.60 a	3.78 b	0.9 b	2.66 b	0.05 a
Crab	14.90 a	3.48 c	5.39 a	0.49 b	4.46 a	0.02 a

Values with the same letters are not significantly different according to the DMRT with 95% confidence.

) scaled according to [23] where 0 = No galls; 1 = 1–2 galls; 2 = 3–10 galls; 3 = 11–30 galls; 4 = 31–100 galls; and 5 ≥ 100 galls.

2.6. Experimental Design and Statistical Analysis

The experiment was designed in a complete randomized design with four replicates. Results were analyzed by SAS software version 9.1(SAS Institute Inc., Cary, NC, USA).

3. Results and Discussion

Exoskeleton powder amendments were evaluated for their pathogenicity on root-knot nematode and growth of brinjal.

3.1. Influence of Chitinous Amendments on the Number of Root-Knot Nematode Galls

Nematicidal activity of chitinous amendments was analyzed by the number and size of galls on the root. Amendments of the exoskeleton on the chitin-rich fish industry by-product of M. incognita inoculated brinjal significantly (p < 0.05), suppressing the root galls compared to the control. Figure 1 illustrates that both shrimp and crab exoskeleton powders have the ability to suppress the root galling by M. incognita which demonstrated that shrimp and crab exoskeletons have efficient nematicidal activity against M. incognita. The exoskeleton powder of the crab (15 ± 0.75) suppressed the root gall formation more significantly than the control (35 ± 2.0). The size of the galls in the shrimp and crab exoskeleton-treated treatments was smaller over the untreated control.

3.2. Influence of Chitinous Amendments on Plant Growth

Results revealed that the application of exoskeleton amendments was statistically significant (p < 0.05) in brinjal, demonstrating shoot height improvement in plants treated with shrimp and crab exoskeleton powders over the untreated control, where plants treated with chitin amendment exhibited higher results than the control. Plants treated with crab exoskeleton powder achieved maximum shoot length (13.67 cm) compared to the plants treated with shrimp exoskeleton powder and the control. Meanwhile, root length was significantly higher in shrimp shells (5.60 cm) than in the control (4.68 cm), but the root length of brinjal in the crab exoskeleton amendment was smaller (3.78 cm) than the control (Table 1). The results discovered that Thirunelvelly-purple brinjal was sensitive to crab exoskeleton chitin amendment.

The brinjal plant amended with crab exoskeleton powder attained maximum fresh shoot weight (5.39 ± 0.36 g) followed by shrimp (3.78 ± 0.29 g) over the untreated control (2.84 ± 0.32 g). The dry shoot weight of brinjal was found to be as high as 4.46 ± 0.36 g when plants were treated with crab exoskeleton powder followed by shrimp (2.66 ± 0.31 g) compared to (1.4 ± 0.48 g) the control. The highest root weight was detected when plants were treated with shrimp exoskeleton powder instead of plants treated with crab exoskeleton powder on both a fresh and dry basis.

This study revealed that the exoskeletons of shrimps and crabs have a significant effect on M. incognita in eggplants. The exoskeleton used in our study showed a different level of nematicidal effect. Among the exoskeleton amendments, crab exoskeleton highly suppressed the extent of gall formation compared to the shrimp exoskeleton and the untreated control, and the sizes of the formed galls were smaller. Chitin induced the soil microorganism population which are actinomycetes, bacteria and a limited number of fungal species containing chitinolytic properties since the nematode’s body has chitin materials. The chitinolytic properties degrade the nematode eggshell chitin, increase chitinase enzyme activity and active defense mechanisms. In chitinolytic fungi, chitinase activity causes pressure within the egg cuticle which causes the penetration and degradation of egg [4,24]. This microbial environment is a pitfall in M. incognita [21], as a significant reduction in the number of galls and nematode eggs occurs [25].

These exoskeleton amendments not only inhibit gall formation but also induce plant growth performance. Crab and shrimp exoskeleton powders stimulated the brinjal shoot height as well as shoot weight on a fresh and dry basis. Shoot dry matter contents increased when chitosan was applied [24,26]. Within the brinjal amended with crab powder, maximum shoot growth was exhibited. In the case of the root growth of brinjal, plants treated with shrimp exoskeleton powder achieved the highest root length, meanwhile the root weight was smaller compared to the control after one month. Crab exoskeleton powder induced the shoot growth while highly suppressing the root growth performance. Hence, the root weights of plants treated with crab exoskeleton powder were significantly lower compared to other treatments. Previous studies reported that ammonia is released through chitin decomposition. Ammonia is toxic to roots and nematodes [4].

4. Conclusions

Shrimp shell and crab chitin materials reduced the number and size of root-knot formation in brinjal and induced plant growth and biomass accumulation. The number of galls formed in crab chitin material treatments was significantly low (15.5), whereas the root length was high (5.60 cm). Chitin materials play a dual role as organic amendments, increasing production and reducing the nematode incidence. Chitin materials are abandoned fish industry wastes in Sri Lanka; therefore, farmers can use these materials as an eco-friendly alternate to synthetic hazardous nematicides in organic agriculture.