Management of Root-Knot Nematode (Meloidogyne incognita) in Okra (Abelmoschus esculentus L.) Moench) using Trichoderma Species and Poultry Manure

Daniel Offiong Etim1*, Idorenyin Asukwo Udo2, Rosemary Anietie Bassey1, Victoria Barrong Ogar1 and Etim John Umana1

1Department of Botany, Faculty of Biological Sciences, University of Calabar, Calabar, Nigeria; 2Department of Crop Science, Faculty of Forestry and wildlife Resources Management, University of Calabar, Calabar, Nigeria.

Received | November 06, 2023; Accepted | December 07, 2023; Published | December 26, 2023

*Correspondence | D.O. Etim, Department of Botany, Faculty of Biological Sciences, University of Calabar, Calabar, Nigeria; Email: [email protected]

Citation | Etim, D.O., Udo, I.A., Bassey, R.A., Ogar, V.B. and Umana, E.J., 2023. Management of root-knot nematode (Meloidogyne incognita) in okra (Abelmoschus esculentus L.) Moench) using trichoderma species and poultry manure. Pakistan Journal of Nematology, 41(2): 182-194.

DOI | https://dx.doi.org/10.17582/journal.pjn/2023/41.2.182.194

Keywords | Meloidogyne incognita, Okra, Poultry manure, Trichoderma species, Biocontrol

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Okra (Abelmoschus esculentus (L.) Moench) is a widely cultivated vegetable crop worldwide and very important in the diet of most Africans. It belongs to the family of Malvaceae (Omotoso, 2008). In Nigeria, it is widely grown, distributed and consumed in either fresh or in dried forms (Farinde, 2007). The West African region accounts for more than 75% of okra produced in Africa, but the average productivity in the region is very low (2.5t/ha) compared to East Africa (6.2t/ha) and North Africa (8.8t/ha) (FAOSTAT, 2006). Nigeria is the second largest producer of okra in the world after India and largest producer in Africa (5.8 million tons), followed by Cote d’ Ivoire, Ghana and then others (FAOSTAT, 2011). Okra provides an important source of vitamins, calcium, potassium, carbohydrates and protein in large quantities which are important in disease prevention (Adebayo and Oputa, 1996). The essential amino acids that okra contains are comparable to that of soybean making it a crucial part of human diet. The plant has immense medical and industrial values (Osawuru et al., 2011). Alcohol extract from the leaves can eliminate oxygen free radicals, improve renal function and reduced proteinuria (Siemonsma and Hamon, 2002). It has also been reported to prevent cancer and heart disease (Idu, 2009) as well as mucilage in mid-wifery (Obiere, 2002). Okra helps in lowering the blood sugar level by blocking the absorption of sugar in the intestinal tract.

The cultivation of okra is limited by biotic and abiotic stress factors. The poor yield of okra in Nigeria has been attributed in part to root knot disease caused by Meloidogyne species. The major limiting factor for its cultivation is the incidence of yellow vein mosaic virus which is transmitted by white fly. Fruits are mostly yellow, tough, small and fibrous once infected by this virus. Also, a number of insect pests like Alphes gossypie, Thrips tabici and Helicoverpa armigera are the deadliest major insect pests of okra (Osawuru et al., 2011). In order to improve the performance and yield of okra, there is need to find ways to combat these emerging pests through the use of different control measures such as biological, chemical and cultural methods. Although, chemical control of nematode pest remains the most effective control measure, there are some serious constraints. Chemical nematicides are very toxic to mammals and beneficial soil microfauna, pollute ground water and have residual effect on farm produce (Lohar, 2001). The use organic manure (poultry manure) and fungi (Trichoderma species) as a component of integrated nematode management is fast gaining wide acceptance (Faruk, 2019).

There are a number of approaches aimed at controlling root-knot nematodes through application of nematicides (Udo et al., 2014). Organic soil amendments from poultry droppings (Hussain et al., 2011), cultural management, physical and biological measures (Giannakou et al., 2019) have been advocated. Presently, researchers have diverted their attention to manage plant nematodes through the use of organic amendments and biocontrol agents such as fungi (Rao et al., 1997). On this note, the present study evaluated the effect of poultry manure and Trichoderma species on root-knot disease on okra.

Materials and Methods

Experimental site and source of material

This experiment was conducted in the screen house of the Department of Botany, University of Calabar. Okra seeds (Clemson spineless) VGTH-014K (Premier brand seeds) were obtained from National Institute for Horticultural Research (NIHORT) Ibadan, Oyo State. Poultry manure from Battery Cage System was obtained from Real Point Farm, Ikot Offiong Ambai, Akpabuyo Local Government Area, Cross River State.

Soil analysis for physicochemical properties and pre-nematode density

The composite soil sample (0-15cm) depth was subjected to routine soil analysis and also nematode population density was examined in the Soil Science Teaching and Research Laboratory, Department of Soil Science, University of Calabar.

Building up of nematode population/inoculum preparation

A pure stock culture of M. incognita was multiplied on susceptible Indian spinach (Basella rubra) plants in the screen house in a steam-sterilized sandy-loam soil. The nematode population was considerably built up at full maturity of the Indian spinach plants. The plants were slightly watered to soften the soil and thus allow for easy uprooting of the plants. The shoot system above the heavily galled root system was cut off and galled root properly washed with water to remove adhering soil particles. The gall root was then placed in moisten polythene bag and taken to the laboratory for inoculation preparation. The heavily galled roots of the Indian spinach plants were uprooted 8 weeks after planting and washed clean with tap water. The galled roots were cut into 1-2 cm segments for larvae extraction. The galled roots were then placed in a blender and distilled water added, blended at 5 seconds interval. The blended substrate was emptied into 1000 ml beaker and more water added and stirred. The suspension of 30 ml was poured into a nematode counting dish and the number of the larvae was counted with the use of a stereomicroscope. An average of such five counts gave approximately 5000 second stage juveniles per 30 ml of solution which was the inoculum density required.

Collection and culturing of Trichoderma species

The soil around the Piggery Farm of Faculty of Agriculture University of Calabar, Calabar and Zoo Garden Mary Slessor Road, Calabar were collected using polyethylene bags, laboratory gloves, masking tape and trowel.

Sterilization of materials

The collected soil sample was taken to the laboratory in a sterilized polyethylene bags. The laboratory work bench was sterilized using sodium hypochlorite (bleach) solution and 99% of ethanol. The sterilization method used in this work was the wet steam method with an autoclave set at 160oC for 30 minutes.

Preparation of media for culturing of Trichoderma species

Potato Dextrose Agar (PDA) was used for culturing the fungus. The measured quantity of PDA (39 g) was poured into 1000 ml conical flask. 1000 ml of distilled water was added and stirred using magnetic stirrer until the solution gave a paste. Nonabsorbent cotton wool was covered with aluminum foil and used to cork the mouth of the conical flask which was rapped again with aluminum foil up to the neck of the flask. The medium was placed in an autoclave at 121 degrees Celsius for 15 minutes. The medium was allowed to cool before removing from the autoclave then 500 mg of Chloramphenicol was added to the solution and allowed to cool before pouring into labeled sterile Petri dishes.

Isolation of Trichoderma species

Soil samples collected were dropped in the plates containing PDA solution and labeled accordingly. The plates were incubated at room temperature of 27±1oC and daily observations were made for emergence of fungal colonies. Colonies formed were subculture to obtain pure cultures of the isolate.

Identification of the Trichoderma species

A drop of Lacto phenol in cotton blue was used to stain the slide. Sterilized inoculation needle was used to pick the spores of the fungi from culture plates and placed on the slide containing the lacto phenol in cotton blue then covered with cover slide for observation and identification under a light microscope (Olympus Optical Philippines) with magnification (x40). The morphological structures of the fungi were compared with those in the Atlas of Imperfect Fungi by Barnett and Hunter (1998) for identification.

Serial dilution of Trichoderma species

Sodium chloride (NaCl) of 0.85g was measured using Ohaus sensitive weighing balance and dissolved in 100 ml of distill water. The solution of (1ml) sodium chloride (NaCl) was diluted in (99ml) of distill water to give one percent (1%) sodium chloride (NaCl) solution in 250ml conical flask. This preparation was used as normal saline. A wire- loop (5 ml in diameter) of the Trichoderma species obtained from a 4-days old pure culture colony of fungi stock was diluted in one milliliter of one percent normal saline solution in 250 ml conical flask. Four test tubes in a test tube rack were labeled and arranged on the laboratory bench. The 9ml of normal saline was added to all test tubes. Automatic pipette was used to draw out one ml of the stock solution and added to the first test tube and mixed thoroughly using vortex mixer for one minute to have homogenous mixture which was the dilution concentration of 10-4 spores/ml of distilled water.

Another 1ml was transferred from the homogenous mixture of the first test tube into the second test tube and mix thoroughly to get dilution concentration of 10-6 spores/ml. This process was repeated for the remaining test tubes to get the dilution concentration of 10-8 spores/ml and 10-10 spores/ml respectively. One milliliter of the dilution solution of the last test tube was pipetted and discarded. This serial dilution process was done separately for the two species of Trichoderma.

Spore count of Trichoderma species

Spore count was done according to Pitt and Poole (1981) where a single chamber of hemacytometer (Neubabuer chamber) was used for the count. The spore concentration was determined as millions of spores per ml of sample and the number of spores generated was used for inoculation into bottles according to each treatment. The spores counted were as follows:

Trichoderma harzianum (T.h) 1×10-4 gave 2.40×107 spores/ ml

Trichoderma harzianum (T.h) 1×10-6 gave 1.40×107 spores/ml

Trichoderma harzianum (T.h) 1×10-8 gave 1.10×107 spores/ml

Trichoderma harzianum (T.h) 1×10-10 gave 9.0×106 spores/ ml

Trichoderma viride(T.v) 1×10-4 gave 2.65× 107 spores/ml

Trichoderma viride(T.v) 1×10-6 gave 2.40×107 spores/ml

Trichoderma viride (T.v) 1×10-8 gave 1.85×107 spores/ml

Trichoderma viride(T.v) 1×10-10 gave 1.05×107 spores/ml

Preparation of carrier for Trichoderma species

Preparation of carrier for Trichoderma species was done according to Sivan et al. (1984). Five grams of millet grain was weighed using sensitive weighing balance. The millet was soaked in water for 24 hours to ferment before used. Normal sterilization of millet grains inside the bottles was done using autoclave at the range of 121oC for 15 minutes. The millet grains in the sterilized Bima bottles were allowed to cool before automatic pipette each indicating for Trichoderma viride and Trichoderma harzianum at different concentrations was used to draw out 1 ml of the spore count solution from the test tubes and inoculate into different designated bema bottle treatments containing sterilized substrates. Filter paper was used to cover the mouth of the bema bottles and the bored holes of the cover bottles were used to seal the mouth after inoculation. The substrates were taken immediately to screen house for inoculation into pots assigned treatments.

Poultry manure treatment, application and planting

Poultry manure was cured by using the in- house manure drying system on Slats method according to Elson and King (1975). The manure was kept for two weeks before applying on each pot receiving treatment. Ten tons per hectare (10t/ha), fifteen tons per hectare (15t/ha) and twenty tons per hectare (20t/ha) of poultry manure was used as the rate of application equivalent to 30, 50, and 60 grams per pot, respectively. Each pot was sown with three seeds of okra in 5kg of sterilized loamy sand and later thinned to one plant stand per pot at two weeks after planting (2WAP).

Nematode inoculation of potted okra plants

The pots were arranged on the screen house in a completely randomized design. Normal spacing of 30cm between rows and 30cm within rows was maintained. At 2WAP, the soil around the plant was excavated to the depth of 5cm and nematode suspension of 30ml (5,000 infective larvae) was inoculated into the soil. The soil around the roots was covered immediately.

Inoculation of potted okra plant using spores of Trichoderma species

The prepared millet grains carrier of 5 grams in Bima bottles having the inoculated Spores of Trichoderma harzianum and Trichoderma viride were taken to screenhouse for inoculation into pots. Holes were made in the soil some distance away from the okra plant. Inoculation of both species of Trichoderma was done at different spore concentrations in all the okra pots receiving treatments except the un-inoculated ones which served as control. The inoculated area was covered with soil immediately after inoculation.

Experimental layout, data collection and statistical analysis

The experiment was laid down in completely randomized design (CRD) having thirty-six treatments and three replications. Data were collected at two weeks interval starting from three weeks after planting (3WAP) on the following: Number of nematodes per plant, number of galls per root system, gall index, nematode reproduction factor, nematode density in 5kg of soil, plant height, number of leaves per plant, days to flowering, photosynthetically active radiation per plant, number of pods per plant, fresh pod weight per plant, mean weight of pod, fresh root weight per plant, dry leaf weight per plant and dry stem weight per plant. Data collected were subjected to analysis of variance (ANOVA) and significant means were separated using Duncan New Multiple Range Test (DNMRT) at 5% probability level.

Results and Discussion

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on gall index (GI) of okra plants inoculated with Meloidogyne incognita

Table 1 shows okra plant (Clemson spineless) was highly susceptible to Meloidogyne incognita infection in pots without poultry manure as the gall index was rated 5.00. Poultry manure (PM) amendment generally reduced the susceptibility rating of okra plants from highly susceptible (G.I= 5.00) to susceptible (G.I= 4.00). Infestation of Trichoderma viride alone at the highest spore concentration of 2.65×107 spores/ ml (V1) reduced the susceptibility rating to moderately susceptible (G.I= 3.00) and this was the trend when both species of Trichoderma were combined with different rates of poultry manure. However, the combination of the highest spore load of either species of Trichoderma 2.40×107 spores/ml (H1+P3) and 2.65×107 spores/ml (V1+P3) with the highest rate of poultry manure 20 t/ha (P3) changed the susceptibility rating (G.I= 2.00).

Table 1 shows increase in poultry manure rate significantly (P≤0.05) increased fresh root weight of okra plants. However, soil infested with 1.85×107 or

 

Table 1: Effects of poultry manure and Trichoderma species on different parameters of okra plant inoculated with Meloidogyne incognita.

Treatment rates/ concentration	Gall index	Plant height (cm)**	Number of leaves	Fresh root weight (g)/plant	Fresh pod weight (g)	Dry leaf weight/ plant(g)	Dry stem weight/ plant (g)
P0 (control)	5.00±0.00a	7.67± 0.333P	3.67±0.333k	11.03± 0.285tu	15.20±1.752t	1.843± 0.0498t	1.543±0.262t
P1 (10 t/ha)	4.00±0.00b	9.33± 0.333mno	5.00±0.00ghi	12.40± 0.364s	34.78±1.044p	3.753± 0.0657q	4.567±0.294p
P2 (15 t/ha)	4.00±0.00b	11.17± 0.167jk	5.67±0.333efg	15.60± 0.666pq	39.87±1.616o	4.107± 0.0698op	5.907±0.295o
P3 (20 t/ha)	4.00±0.00b	12.17± 0.167hi	6.34±0.333cde	18.28± 0.513n	48.23±1.737mn	4.900± 0.0964mn	7.983±0.0376m
H1(2.40×107 spores/ml)	3.67±0.333c	10.17± 0.167l	4.67±0.333hij	14.73± 0.260q	26.67±1.271q	2.213± 0.0441s	2.503±0.227rs
H2(1.40×107 spores/ml)	4.00±0.00b	10.00± 0.00lm	4.34±0.333ijk	12.82± 0.216rs	19.20±1.770rst	2.073± 0.0410st	2.187±0.0876s
H3(1.10×107 spores/ml)	4.00±0.00b	9.00± 0.00no	4.34±0.333ijk	11.02± 0.145tu	19.99±0.948rs	2.043± 0.0406st	1.620±0.193t
H4 (9.0×106 spores/ml)	4.00±0.00b	8.67± 0.333o	4.00±0.00jk	10.24± 0.312u	18.41±0.768rst	1.987± 0.0176st	1.397±0.176t
V1 (2.65×107 spores/ml)	3.00±0.00d	11.33± 0.333jk	5.34±0.333fgh	15.92± 0.174op	29.29±0.616q	2.727± 0.0694r	4.027±0.0561q
V2 (2.40×107 spores/ml)	4.00±0.00b	9.67± 0.333lmn	4.34±0.333ijk	13.62± 0.266r	22.14±1.199r	2.347± 0.0606s	2.967±0.107r
V3 (1.85×107 spores/ml)	4.00±0.00b	9.67± 0.333lmn	4.67±0.333hij	11.82± 0.232st	18.03±0.917rst	2.233± 0.0882s	2.467±0.0726rs
V4 (1.05×107 spores/ml)	4.00±0.00b	8.67± 0.333o	4.00±0.00jk	10.88± 0.0726tu	17.47±0.924st	2.097± 0.0606st	2.160±0.757s
H1+ P1	3.00±0.00d	12.17± 0.167hi	6.00±0.00def	17.73± 0.249n	62.40±1.774k	4.410± 0.105o	9.197±0.318l
H2+ P1	3.00±0.00d	11.17± 0.167jk	5.67±0.333efg	16.63± 0.293op	55.40±1.019l	4.093± 0.0636op	7.987±0.0689m
H3+ P1	4.00±0.00b	11.17± 0.167jk	5.34±0.333fgh	16.73± 0.217o	48.31±1.287mn	3.830± 0.0666pq	6.947±0.0902n
H4+ P1	4.00±0.00b	9.83± 0.167lm	5.34±0.333fgh	15.69± 0.260opq	44.99±1.063n	3.600± 0.0577q	6.313±0.104o
V1+ P1	3.00±0.00d	13.17± 0.167fg	6.67±0.333bcd	21.89± 0.310jk	65.03±2.707k	5.857± 0.148k	10.910±0.121j
V2+ P1	3.00±0.00d	12.17± 0.167hi	6.34±0.333cde	20.72± 0.319lm	60.91±0.914k	5.263± 0.0684l	9.933±0.161k
V3+ P1	3.67±0.333c	11.17± 0.167jk	6.00±0.00def	19.80± 0.156m	51.45±1.077m	4.733± 0.0726n	9.110±0.0985l
V4+ P1	4.00±0.00b	10.83± 0.167k	5.67±0.333efg	25.69± 0.180n	50.38±0.896m	4.233± 0.0882o	8.173±0.0722m
H1+ P2	3.00±0.00d	13.83± 0.167ef	7.00±0.00bc	25.69± 0.641hi	82.41±1.190efg	6.600± 0.176j	13.273±0.241ef
H2+ P2	3.00±0.00d	12.50± 0.289ghi	6.34±0.333cde	22.14± 0.491j	74.27±1.730ij	5.923± 0.0788k	12.007±0.0762h
H3+ P2	3.00±0.00d	11.83± 0.167ij	6.00±0.00def	20.94± 0.362kl	71.24±0.927j	5.400± 0.153l	11.033±0.142ij
H4+ P2	3.00±0.00d	11.17± 0.167jk	6.00±0.00def	19.87± 0.164m	64.07±2.128k	5.083± 0.0726lm	10.143±0.0536k
V1+ P2	3.00±0.00d	15.33± 0.333c	7.34±0.333b	31.63± 0.895e	92.72±2.075b	8.580± 0.191ef	15.047±0.111d
V2+ P2	3.00±0.00d	14.17± 0.167de	6.67±0.333bcd	27.88± 0.415g	86.46±0.575de	8.040± 0.0569g	13.570±0.229e
V3+ P2	3.00±0.00d	14.17± 0.167de	6.34±0.333cde	26.22± 0.383h	79.13±1.006gh	7.267± 0.120hi	12.663±0.0984g
V4+ P2	3.00±0.00d	12.83± 0.167gh	6.34±0.333cde	25.03± 0.120i	71.05±0.875j	6.967± 0.0751i	11.433±0.203i
H1+ P3	2.00±0.00f	16.17± 0.167b	7.34±0.333b	32.10± 0.276e	92.13±1.460bc	9.387± 0.178c	15.663±0.235c
H2+ P3	2.33±0.333e	15.17± 0.167c	6.67±0.333bcd	30.07± 0.105f	85.04±0.729def	8.777± 0.0784de	14.887±0.0928d
H3+ P3	3.00±0.00d	14.83± 0.167cd	6.34±0.333cde	28.46± 0.219g	81.99±0.878fg	8.023± 0.0536g	13.540±0.155e
H4+ P3	3.00±0.00d	14.33± 0.333de	6.00±0.00def	27.55± 0.318g	76.46±1.168hi	7.433± 0.117h	12.823±0.184fg
V1+ P3	2.00±0.00f	17.50± 0.289a	8.34±0.333a	39.50± 0.454a	106.43±2.470a	10.787± 0.368a	17.590±0.135a
V2+ P3	2.33±0.333e	16.17± 0.167b	7.34±0.333b	36.74± 0.321b	95.98±1.271b	9.873± 0.153b	16.283±0.249b
V3+ P3	3.00±0.00d	14.33± 0.167de	6.67±0.333bcd	35.34± 0.285c	88.33±1.448cd	9.030± 0.0361d	14.857±0.0606d
V4+ P3	3.00±0.00d	13.67± 0.333ef	6.34±0.333cde	33.90± 0.103d	80.41±0.739gh	8.333± 0.136fg	13.153±0.264efg

 

*Means within a column followed by the same letter of alphabet are not significantly different from one another based on Duncan’s New Multiple Range Test (DNMRT) at 5% probability level. **Plant height; three weeks after planting (3WAP), P= poultry manure; V= Trichoderma viride; H = Trichoderma harzianum.

 

1.05×107 spores/ml (V3 or V4) of Trichoderma viride and 1.10×107 or 9.0×106 Spores/ml (H3 or H4) of Trichoderma harzianum species produced okra plants that had similar fresh root weight with the control plants (Po). Increase in the spore load of both Trichoderma species led to a significant increase in fresh root weight. The combination of either species of Trichoderma at a given spore load with poultry manure at a specified rate produced okra plants with significantly higher fresh root weight than individual control agent at that given rate or spore load. The combination of all spore concentrations 2.65×107 to 1.05×107 spores/ml (V1+P3 to V4+P3) of Trichoderma viride with 20 t/ha poultry manure (P3) produced plants with significantly (P≤0.05) heavier fresh roots than the other treatments. However, (V1+P3) Trichoderma viride at 2.65×107 spores/ml in combination with 20 t/ha poultry manure (P3) produced okra plants with the heaviest fresh root weight.

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on the plant height (cm) of okra inoculated with Meloidogyne incognita

Table 1 shows that at three weeks after planting (3WAP), poultry manure amendment and inoculation of either species of Trichoderma significantly (P≤0.05) enhanced the growth of okra plants compared with the control. Increase in the application rate of poultry manure led to a corresponding significant (P≤0.05) increase in height. In most cases, there was no significant (P>0.05) difference in height between plants inoculated with the two species of Trichoderma at a given spore load. However, there was a significant enhancement in okra plant height when poultry manure was combined with either species of Trichoderma compared with individual application of the control agents at given rate of poultry manure application or inoculum load of Trichoderma species. The results at 5 and 7 WAP followed the trend of 3WAP. However, at 5WAP, plants inoculated with 9.0×106 Spores/ml of Trichoderma harzianum (H4) were similar in height with the control plants (P0) and the same was true for those inoculated with 1.10×107 spores/ml (H3) and 9.0 ×106 Spores/ml Trichoderma harzianum (H4) and 1.05 ×107 Spores/ml of Trichoderma viride (V4). At all sampling periods, inoculation with 2.65 × 107 Spores/ml of Trichoderma viride (V1+P3) in combination with 20 t/ha (P3) of poultry manure amendment produced significantly (P≤0.05) the tallest plants.

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on the number of leaves of okra inoculated with Meloidogyne incognita

Table 1 shows that at three weeks after planting (3WAP), application of poultry manure significantly (P≤0.05) enhanced leaf production in okra plants compared with the unamended control (P0). In contrast, infestations of soil with lower inoculum load of 1.85×107 to 1.05×107 Spores/ml (V3 to V4) of Trichoderma viride and of Trichoderma harzianum 1.10×107 to 9.0×106 spores/ml (H3 to H4) did not (P>0.05) enhance leaf production compared with the uninfected soil. The combination of either species of Trichoderma with successive increase in the rate of poultry manure amendment significantly increased the number of leaves produced when compared with individual application of each control agent. At 5 and 7WAP, higher rates of poultry manure (15t /ha and 20 t/ha) significantly enhanced leaf production compared with the control. Infestation of soil with either species of Trichoderma alone irrespective of the inoculum load produced plants with similar or less number of leaves relative to the control. However, the combination of the Trichoderma species with 15 t/ha (P2) or 20 t/ha (P3) of poultry manure led to a significant enhancement in leaf production compared with the control plants. Infestation of soil with Trichoderma viride at 2.65×107 spores/ml (VI+P3) in combination with 20 t/ha (P3) of poultry manure significantly produced plants with the highest number of leaves closely followed by Trichoderma harzianum at 2.40×107 Spores/ml (HI+P3) in combination with the highest rate of poultry manure.

The effects of poultry manure (PM) amendment and soil infestation with Trichoderma species on the leaf and stem dry weights of okra plants inoculated with Meloidogyne incognita

Table 1 shows that with the exception of lower inoculum load of both species of Trichoderma, all the treatments significantly (P≤0.05) enhanced leaf dry matter accumulation in okra plants relative to the control. Successive increase in the poultry manure rate significantly (P≤0.05) increased leaf dry weight. The combination of poultry manure with either species of Trichoderma consistently increased leaf dry weight of okra compared with individual application of the control agents at a given rate. However, Trichoderma viride at 2.65×107 Spores/ml (V1) in combination with 20 t/ha poultry manure (P3) produced plants with significantly the highest leaf dry matter. Results of the effects of treatments on dry stem weight followed the trend of leaf dry weight. However, Trichoderma viride infestation at all inoculum loads significantly (P≤0.05) enhanced stem dry matter accumulation compared with control. Trichoderma viride at 2.65×107 Spores/ml (V1+P3) in combination with 20 t/ha poultry manure significantly (P≤0.05) produced okra plants with the highest stem dry weight.

The effects of poultry manure (PM) amendment and Trichoderma species inoculation on fresh pod weight (g) of okra inoculated with Meloidogyne incognita

Table 1 shows increase in poultry manure rate significantly (P≤0.05) increased okra fresh fruit yield compared with the control. Infestation of soil with highest spore load of either species of Trichoderma alone (H1 and H2) significantly (P≤0.05) enhanced fresh fruit yield of okra plants relative to the control, while lower spore load produced similar fresh fruit yield as the control. Combinations of poultry manure at various rates with the different spore load of either species of Trichoderma led to a significant (P≤0.05) fresh fruit yield increase compared with individual application at the specified rates of poultry manure or spore load of Trichoderma specie. However, the combination of the highest rate of poultry manure 20 t/ha (P3) with the highest spore load of Trichoderma viride (V1+P3) produced plants with the highest fresh fruit yield (P≤0.05). There were no significant (P>0.05) differences in fresh fruit yield among plants treated with Trichoderma viride at 2.65×107 spores (V1) in combination with 15 t/ha poultry manure (P2), Trichoderma viride 2.65×107spores/ml (V1) and Trichoderma harzianum at 2.40×107 spores/ml (H1) both in combination with 20 t/ha poultry manure (P3).

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on number of galls per root system of okra plants inoculated with Meloidogyne incognita

Figure 1 shows that soil amendment with poultry manure significantly (P≤0.05) reduced root galling by the nematode species with the highest rate of poultry manure P3 (20t/ha) recording the least number of galls. Soil infestation with both species of Trichoderma with the least inoculum load 1.10×107 to 9.0×106 spores/ml (H3 to H4) and 1.85×107 to 1.05×107 spores/ml (V3 to V4) recorded okra stands with significantly (P≤0.05) fewer galls than poultry manure amendment with the highest rate P3(20 t/ha). The increase in inoculum load for both species of Trichoderma resulted in a significant (P≤0.05) increase in root galls comparatively. The highest spore concentration of Trichoderma viride (V1) had plants with significantly fewer numbers of galls than that of Trichoderma harzianum (H1). The combination of poultry manure with Trichoderma species always resulted in a significant (P≤0.05) decrease in the number of galls except in the case of 1.10×107 spores/ml with 10t/ha (H3+P1), 9.0×106 spores/ml with 10t/ha (H4+P1) and 1.05×107 spores/ml with 10t/ha (V4+P1). However, both Trichoderma species at their highest spore concentrations in combination with highest rate 20 t/ha of poultry manure (P3) produced okra plants with the least number of galls.

 

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on number of nematodes per plants inoculated with Meloidogyne incognita

Figure 2 shows that nematode density per okra plant was significantly (P≤0.05) reduced with increase in the poultry manure (PM) rates and spore concentration of both Trichoderma species compared with the control. However, at each spore load, Trichoderma viride 2.65×107 to 1.05×107 spores/ml (V1 to V4) gave a lower nematode density (P≤0.05) relative to Trichoderma harzianum 2.40×107 to 9.0×106 spores/ml (H1 to H4) infested plants. The combination of either species of Trichoderma with Poultry manure always produced plants with significantly (P≤0.05) lesser number of nematodes compared with individual application rates (control). However, plants infested with Trichoderma viride at spore load of 2.65×107 spores/ml (V1+P3) in combination with the highest poultry manure rate of 20 t/ha (P3) produced the least number of nematodes (P≤0.05) closely followed by the same species at 2.40×107 spores/ ml (V2+P3) at the same poultry manure rate 20 t/ha (P3) and then Trichoderma harzianum at 2.40×107 spores/ml (H1+P3) in combination with 20 t/ha (P3) poultry manure.

 

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on nematode density in soil inoculated with Meloidogyne incognita

Figure 3 shows that the nematode population in the soil after harvest almost followed the trend of that in the plant. With the exception of 10 t/ha poultry manure rate, poultry manure amendment significantly (P≤0.05) reduced nematode number in the soil compared with the control experiment. Similarly, infestation of okra plants with both Trichoderma species alone significantly reduced soil nematode population. However, in both species of Trichoderma, increase in inoculum load significantly (P≤0.05) reduced nematode number in the soil. Trichoderma viride significantly reduced more number of nematodes in the soil than Trichoderma harzianum at each respective inoculum load. Combination of poultry manure with either species of Trichoderma resulted in a significant (P≤0.05) decrease in soil nematode population when compared with individual control agent at any given rate of application. However, the least number of nematodes in the soil was obtained when the soil was amended with 20 t/ha poultry manure and infested with 2.65×107 spores/ml of Trichoderma viride (V1+P3), Trichoderma harzianum at 2.40×107 spores/ml (H1+P3) combined with 20 t/ha poultry manure gave the same number of nematodes in the soil as Trichoderma viride at 2.40×107 spores/ml combined with 20 t/ha poultry manure amendment.

 

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on nematode reproduction factor (Rf) inoculated with Meloidogyne incognita

Figure 4 shows the nematode reproduction factor (Rf) was significantly (P≤0.05) higher in the control experiment (no poultry manure amendment and no Trichoderma species inoculation), 10 t/ha poultry manure application and Trichoderma harzianum at 9.0×106 spores/ml (H4) inoculation than other treatments. Higher rates of poultry manure amendment; 15 t/ha (P2) and 20 t/ha (P3) significantly (P≤0.05) reduced reproduction factor (Rf). Increase in the concentration of spores of both Trichoderma species significantly (P≤0.05) inhibited root-knot nematode reproduction in okra plants. However, at each respective spore load, Trichoderma viride significantly inhibited nematode reproduction more than Trichoderma harzianum. The combination of either species of Trichoderma with poultry manure at a given rate of application was in most cases more efficient (P≤0.05) in impeding nematode reproduction than individual application of the control agents. However, Trichoderma viride at 2.65×107 to 1.85×107 spores/ml (V1+P3 to V3+P3) in combination with 20 t/ha poultry manure and Trichoderma harzianum at 2.40×107 to 1.40×107 (H1+P3 to H3+P3) combined with 20 t/ha poultry manure had the least soil nematode density (P≤0.05).

 

The okra variety (Clemson spineless) used in this study was found to be highly susceptible to Meloidogyne incognita with gall index of 5.00 in soils without poultry manure and uninfested with either species of Trichoderma. This observation is in line with the report of Nwanguma (2002). Severe galling on the roots of okra plants by Meloidogyne incognita could be attributed to a conducive soil environment as nematodes activity is generally enhanced in sandy soils as reported by Agu (2008) and Olewe (2005). Increase in the rate of poultry manure led to a significant reduction in root galls, nematode density in both soil and okra plant and the reproduction factor (Rf) compared with soils without amendment. Generally, application of organic amendments either of plant or animal origin has been reported by many authors to suppress nematode population in the soil as well as their damage potentials (Nwanguma, 2002; Radwan et al., 2007; Oka, 2010; Renco, 2013). Various mechanisms have been advanced for the possible effects of organic amendments on the tripartite interaction of nematode–plant soil system (Oka, 2010; Mcsorley, 2011; Renco, 2013). The release of preexisting nematicidal constituents, generation of nematicidal compounds such as organic acids, ammonia, nitrogenous compounds during decomposition, enhancement or stimulation of nematode antagonist, increase in plant resistance/ induced tolerance by microorganisms or natural compounds and changes in the physicochemical properties of the soil are some of the possible mechanisms listed by these authors. However, it could be very difficult to distinguish the most important mechanisms as they may operate simultaneously (Akhtar and Malik, 2000). It is possible that some of these mechanisms could have been involved in the suppression of Meloidogyne incognita population in this study. The poultry manure used in this work had a very narrow C: N of 8.95. Agu (2007) had observed that organic materials with low C: N ratios and high protein or amine type of nitrogen content were more effective in nematode suppression than those with high C: N ratios. In this study therefore, nematodes could have been killed through the release of nitrous acid which is reported to be more potent in acidic soils than ammonia as a product of decomposition (Oka, 2010). The highest nematode suppression was obtained with 20 t/ha poultry manure amendment which corroborates with the work of Nwanguma and Awoderu (2002) who reported high nematicidal activity of chicken litters against Meloidogyne incognita at 16 t/ha amendment rate comparable to a synthetic nematicide mocap.

 

Effects of poultry manure (PM) amendment and Trichoderma species inoculation on number of pods/ plant inoculated with Meloidogyne incognita.

Figure 5 shows that amendment of soil with poultry manure significantly (P≤0.05) enhanced fruit setting by okra when compared with the control experiment. With the exception of the highest spore load of Trichoderma viride, infestation of soil with either species of Trichoderma alone did not significantly (P>0.05) increase the number of pods produced by okra plants relative to the control. In most cases, the combined application of poultry manure and infestation with Trichoderma produced plants that significantly (P≤0.05) had more number of pods than individual application of the control agents. However, the highest spore load (2.65×107 Spores/ml) of Trichoderma virde in combination with the highest rate 20 t/ha of poultry manure (V1+P3) produced plants that significantly (P≤0.05) had the highest number of fruits followed by the highest spore load (2.40×107 spores/ml) of Trichoderma harzianum at the same poultry manure rate (H1+P3) as well as Trichoderma viride at 2.40×107 spores in combination with 20 t/ha poultry manure (V2+P3).

Infestation of soil with Trichoderma harzianum or Trichoderma viride alone significantly inhibited root galling, nematode density in both soil and okra with a decrease in nematode reproduction factor. The highest spore density was the most potent for both species of Trichoderma. These findings validate the work of previous researchers (Windham et al., 1989; Sharon et al., 2001; Sahebani and Hadavi, 2008). Similarly, Spiegel and Chet (1998) reported that the application different isolates of Trichoderma harzianum and Trichoderma lignorum caused significant reduction in Meloidogyne javanica population and root galling and enhanced the growth of infested plants in Israel.

Different mechanisms of biocontrol activity of Trichoderma species against many soil borne pathogens have been suggested which include; antibiosis, competition, mycoparasitism, enzymatic hydrolysis and induced systemic resistance (Sahebari and Hadavi, 2008). Penetration of eggs and colonization of Meloidogyne javanica juveniles have been illustrated in vitro by Sharon et al. (2001). Khan and Saxena (1997) illustrated the nematicidal/nematotoxic properties of the metabolites of Trichoderma viride used as root dip treatment on tomato against Meloidogyne javanica where nematode penetration and reproduction were adversely affected. Sharon et al. (2001) demonstrated the involvement of proteinase in the parasitic behavior of Trichoderma species on Meloidogyne javanica and they suggested that improvement in the proteolytic activity of the antagonist may be very vital for the biological control of plant parasitic nematodes. It is likely that one or more of the mechanisms mentioned above could have been deployed by the Trichoderma species used in this study to suppress the population of Meloidogyne incognita on okra plants with, Trichoderma viride being more efficacious in reducing root galling and nematode reproduction than Trichoderma harzianum. This may partially be attributed to its ability to sporulate faster as observed during culturing. It may suggests that T. viride is capable of exploiting the substrate used as carrier more than T. harzianum for its higher suppression activity on Meloidogyne incognita population. It could also be that its proteolytic activity is more than that of T. harzianum as suggested by Sharon et al. (2001).

The combination of either species of Trichoderma with poultry manure was more potent in reducing root galling and suppressing nematode population than individual application of the nematode control agents. This observation confirms the findings of earlier workers (Ehteshamul-Haque et al., 1996; Rao et al., 1998; El-Sherif and Ismail, 2009). Soil amendments with organic matter have been reported to simulate soil food webs, thus increasing the population of free-living nematodes, plant growth promoting rhizobacteria, fungi and nematode antagonist (Oka, 2010; Mcsorley, 2011). The role of the organic amendment could be complementary as it may help the antagonist like Trichoderma species to establish in the soil. The highest root gall reduction and suppression of Meloidogyne incognita population was obtained with the highest spore density of Trichoderma viride (2.65×107 spores/ml) and Trichoderma harzianum (2.40×107 Spores/ml) in combination with the highest rate of poultry manure (20 t/ha) indicating that the combination of various mechanisms of nematode control by the two agents may likely account for this observation. Amendment of soil with poultry manure and its combination with various spore densities of both species of Trichoderma significantly enhanced growth, dry matter accumulation, photosynthetic rate, earliness to anthesis and fruit yield of okra. These observations could be attributed to the reduction in root galling as against severe galling obtained in soils not amended with poultry manure or infested with Trichoderma species. Root galling by Meloidogyne incognita is reported to impair water and nutrient uptake, translocation and photosynthesis (Williamson and Gleason, 2003). The poultry manure could have acted as a reservoir of plant nutrients slowly released to okra plants during its growth phase and fruiting stage, as the soil used for the trial was deficient in major plant nutrients. Udo and Ugwuoke (2010) reported increased growth and yield of turmeric (Curcuma longa L.) infected with Meloidogyne incognita by poultry manure amendment at the rate of 20 t/ha. They cautioned against higher rate greater than 20 t/ha to be phytotoxic. In the present study, Phytotoxicity was not observed at the highest rate of poultry manure (20 t/ha). Thus, the highest Meloidogyne incognita population suppression, growth and yield enhancement of okra was obtained with the highest spore densities of both species of Trichoderma in combination with amendment of poultry manure at 20 t/ha.

Conclusions and Recommendations

A comparative study between different rates of poultry manure and different spore load of Trichoderma species showed that increase in poultry manure rates and spore load of Trichoderma applied singly significantly reduced root galling, nematode density in soil per pot and nematode reproduction factor. Also, the combination of different rates of poultry manure with either species of Trichoderma at various spore loads significantly inhibited root galling and nematode population more than single application of each control agent.

Acknowledgement

The authors are grateful to Mr. Ekeng Okon Ita of the Cross River State University of Technology, Nigeria for assisting in the isolation of Trichoderma species used in this study.

Novelty Statement

The study has brought to limelight the possibility of using indigenous isolates of Trichoderma viride and Trichoderma harzianum grown on a common agricultural produce (millet) in the management of Meloidogyne incognita.

Author’s Contribution

Daniel Offiong Etim: Designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of manuscript.

Idorenyin Asukwo Udo and Etim Johnson Umana: Managed the analysis of the study.

Rosemary Anietie Bassey and Victoria Barrong Ogar: Managed the literature searches. All authors read and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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