Scholarly article on topic 'Influence of Arbuscular mycorrhiza fungi (AMF) on drought tolerance and charcoal rot disease of cowpea'

Influence of Arbuscular mycorrhiza fungi (AMF) on drought tolerance and charcoal rot disease of cowpea Academic research paper on "Agriculture, forestry, and fisheries"

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{Cowpea / " Macrophomina phaseolina " / "Arbuscular mycorrhizal fungi" / Drought}

Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — B.O. Oyewole, O.J. Olawuyi, A.C. Odebode, M.A. Abiala

Abstract The influence of Arbuscular mycorrhiza fungi (AMF) (Glomus deserticola and Gigaspora gigantea) were evaluated on drought tolerance and charcoal rot disease of cowpea genotypes: IT90K-277-2, IT84S-2246-4 and IT06K123-1. IT90K-277-2 and IT84S-2246-4 were sown in 3kg of sterilized soil for drought experiment with five treatments. Treatment was established thirty days after germination with inoculation of G. deserticola, the mycorrhizal treated cowpea withstand the water stress and produced high yield. Biocontrol experiment had 2kg sterilized soil potted into bags with cultivars IT90K-277-2 and IT06K123-1, fourteen treatments were established with soil drenched before planting and simultaneous inoculation. Soil drenched with AMF before planting and inoculation of M. phaseolina after 10days of germination recorded higher growth parameters, while the simultaneous inoculated plant was the most effective in reducing disease severity. However, simultaneous treatment of G. deserticola, G. gigantea and M. phaseolina were most effective for both growth parameters and reduction of disease severity.

Academic research paper on topic "Influence of Arbuscular mycorrhiza fungi (AMF) on drought tolerance and charcoal rot disease of cowpea"

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Biotechnology Reports

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Influence of Arbuscular mycorrhiza fungi (AMF) on drought tolerance and charcoal rot disease of cowpea ^

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B.O. Oyewolea'b'*, O.J. Olawuyib, A.C. Odebodeb, M.A. Abialac

a National Horticultural Research Institute, Ibadan, Nigeria b Department of Botany, University of Ibadan, Nigeria

c Department of Biological Sciences, College of Basic and Applied Sciences, Mountain Top University, Prayer City, Nigeria

ARTICLE INFO

ABSTRACT

Article history:

Received 31 May 2016

Received in revised form 9 January 2017

Accepted 23 February 2017

Available online 28 February 2017

Keywords: Cowpea

Macrophomina phaseolina Arbuscular mycorrhizal fungi Drought

The influence of Arbuscular mycorrhiza fungi (AMF) (Glomus deserticola and Gigaspora gigantea) were evaluated on drought tolerance and charcoal rot disease of cowpea genotypes: IT90K-277-2, IT84S-2246-

4 and IT06K123-1. IT90K-277-2 and IT84S-2246-4 were sown in 3 kg of sterilized soil for drought experiment with five treatments. Treatment was established thirty days after germination with inoculation of G. deserticola, the mycorrhizal treated cowpea withstand the water stress and produced high yield. Biocontrol experiment had 2 kg sterilized soil potted into bags with cultivars IT90K-277-2 and IT06K123-1, fourteen treatments were established with soil drenched before planting and simultaneous inoculation. Soil drenched with AMF before planting and inoculation of M. phaseolina after 10days of germination recorded higher growth parameters, while the simultaneous inoculated plant was the most effective in reducing disease severity. However, simultaneous treatment of G. deserticola, G. gigantea and M. phaseolina were most effective for both growth parameters and reduction of disease severity. © 2017 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Cowpea (Vigna unguiculata L. Walp.) is one of the most important drought-resistant leguminous food crops grown for its foliage and grain in tropical and sub topical Saharan Africa regions of the world particularly in Nigeria [1,2]. There are variations among cowpea genotypes in their drought tolerating potentials [3,4]. Turk et al. [5] cited in Ahmed and Suliaman [6] affirmed that cowpea is highly sensitive to water stress during the flowering and pod-filling stages. Though, there are limited information on the response of the crop to drought at different stages of growth. According to Ajibade and Amusa [7], its cultivation in humid agroecologies of South West Nigeria is also faced with several pests and diseases such as brown blotch, anthracnose , cercospora leaf spot, choaniphora pod rot, false smut and web blight, charcoal rot and sclerotium stem blight of which Macrophomina phaseolina is included. The effect of field diseases has led to reduction in yield of cowpea.

* Corresponding author at: National Horticultural Research Institute, Ibadan, Nigeria.

E-mail addresses: oyeebarry@yahoo.com (B.O. Oyewole), olawuyiodunayo@yahoo.com (O.J. Olawuyi).

Macrophomina phaseolina (Tassi) Goidanich, is one of the most destructive plant pathogens in the tropics and subtropics causing diseases in a wide range of host plant [8,9]. The pathogen was detected in Chile in 1983 in Pinus radiata D., Don nurseries in the Bio-Bio Region In the last few years, dissemination of the pathogen had been detected from the nurseries to the plantations through asymptomatic plants. Mortality of plant would be observed in the first years of the plantation when they are predisposed to conditions such as hydraulic stress and high soil temperatures [10]. M. phaseolina is a saprophyte that survives in the soil due to micro-sclerotinia formation which is pseudoparenchymal tissue masses resistant to adverse environmental conditions [11].

The most successful control strategy for charcoal root rot in forest nurseries was soil fumigated with methyl bromide [10]. Although, some of the problems associated with the application of chemicals include high cost, environmental pollution, breaking up the ecological balance of the soil, as well as the destruction of the ozone layer [12,13]. Biological control has been considered as an alternative selective method to control this disease [14]. Several researchers had focused on antagonist microbes such as Bacillus, Pseudomonas, Streptomyces, Trichoderma, Penicillium, Rhizopus, Aspergillus but there are limited studies on the adoption of mycorrhiza fungi biotechnology as control strategy Olawuyi et al., 2014a,b.

http://dx.doi.org/10.1016/j.btre.2017.02.004

2215-017X/© 2017 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativec0mm0ns.0rg/licenses/by-nc-nd/4.0/).

Mycorrhiza association is a symbiotic non-pathogenic relationship between plant roots and fungal hyphal [15,16]. In this relationship, the fungi obtain carbon compounds and other nutritional requirements from the symbiotic plant roots, and in return, supply the plant with most of the immobile mineral elements such as Nitrogen (N), Phosphorus (P), Potassium (K), Calcuim (Ca), Copper (Cu) and Zinc (Zn) from the soil solution. The importance of mycorrhiza association in both agricultural and ecological systems had earlier been widely recognised [17,15]. The increased plant growth by VAM association is usually due to increased mineral elements uptake by the hyphae from the soil [18], Olawuyi et al., 2012), improved water relations and pest resistance of host plants [19,20], plants tolerance to a variety of abiotic stresses [21], increased resistance to soil pathogens [22,23,24], 2013). Mycorrhizae can also resist drought in many plants under stress conditions therefore the plants infected with VA mycorrhizae are less likely to wilt under drought affected conditions [25,26,27].

Therefore, the study aimed at investigating the influence of AMF (Glomus deserticola) on water stress in cowpea and established the effect of AMF (Glomus deserticola and Gigaspora gigantea) on Macrophomina phaseolina causing charcoal rot in cowpea.

2.4. Morphological identification and characterization of fungi isolates

The identification and classification of these structures were compared to that of Domsch et al. [28] in compendium of soil fungi.

The electronic microscope was used in viewing the prepared slides for the various morphological characters ranging from micro conidia, macro conidia, conidia shape and hyphal arrangement.

2.5. Soil sterilization and seed viability test

The soil (sandy loam soil) used in conducting the research work was collected from the Research farm of the Department of Botany, University of Ibadan. Sterilization of the soil was done using electric soil sterilizer and allowed to cool before packed into pottling bags.

The test for viability was done using the method of, in which 10 seeds were sown per bag. Each variety was sown in triplicates and the percentage germination was obtained from the formulae below;

%germination =

No. of germinated seed Totalno of seed planted

2. Materials and methods

2.1. Experimental location and research design

Planting of cowpea seeds was done at the screen house, while the isolation and identification were carried out at the research farm and pathology laboratory respectively in the Department of Botany, University of Ibadan. complete randomized design was used for the two experiments with three replicates.

2.2. Source of seed samples and inocula

Cowpea seeds were collected from the germplasm unit of the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Seeds varieties include: drought susceptible (1T90K-277-2 and 1T84S-2246-4), and pathogen susceptible (1T06K-123-1 and 1T90K-277-2). Rhizosphere soil of cowpea where the pathogenic organism was isolated, was also collected from 11TA, while Arbuscular mycorhiza fungi (Glomus deserticola and Gigaspora gigantea) were obtained from the Department ofBotany, University of 1badan.

2.3. Media preparation, isolation of organism and slide preparation

20 g of PDA dissolved in 500mls of distilled water was prepared and autoclaved at 121 °C for 15mins.The prepared PDA was allowed to cool to 45 °C, while streptomycin was added to inhibit the growth of bacteria and the solution was gently swirled to obtain a homogeneous mixture. 2 g of soil sample was serially diluted using dilution factors of 10-1,10~2,10~3,10-4,10~5and 10~6,1 ml is taken each from 10_1, 10~3 and 10~6 inoculated into prepared Potato dextrose Agar media (PDA) in Petri dishes using pour plate method, Incubation was done at room temperature for 5-7 days. Mixed culture was sub-cultured to obtain pure culture of the pathogen. Stock culture was obtained from the pure culture of the organism which were prepared on PDA slant and stored at about 4 °C in the refrigerator. Slides of pure cultures obtained were prepared using sterilized needle to pick mycelia growth of the organism onto the slide surface. With the aid of the needle and addition of drop of sterile distilled water, the mycelia were properly dislodged. A drop of lacto phenol in cotton blue stain was then added to aid clarity when viewed under microscope.

2.6. Seed planting and pathogenicity test

A sterilized sandy loam soil weighing 2 kg and 3 kg potted into pottling bags were used for disease resistance and drought tolerance experiments in varieties of cowpea respectively. Ten seeds were planted per bag and then thinned to two per bag after three weeks of growth.

The pathogenicity test was carried out using modified method of Ahmed et al. [51]. The mycelia suspension of Macrophomina phaseolina isolate was prepared by blending ten 5 mm mycelia disc from 10 to 15 day-old culture of the fungus in 100 ml of sterile distilled water using warring blender. 5 ml of the inoculum mycelia suspension was injected round the wounded region of the lower stem of the seedlings after 10 days of germination.

Reactions of cowpea varieties to drought as influenced by Glomus deserticola

Ten seeds each of the drought susceptible cowpea varieties (IT90K-277-2 and IT84S-2246-4) were sown separately in 3 kg sterilized sandy loam soil. Germination of seeds was observed after 4 days of planting. The plants were thinned to two seedlings per plant after 14 days of emergence. Five treatments were established on the two cowpea varieties after 30 days of germination with inoculation of 5 ml mycelia suspension of Macrophomina phaseolina and 10 g (32spores) of Glomus deserticola. These treatments included;

T1- cowpea alone + watering T2- cowpea alone + water stressed

T3- cowpea infested + AMF (Glomus deserticola) + water stressed T4- cowpea infested + AMF (Glomus deserticola) + watering T5- cowpea infested+AMF (Glomus deserticola) + Macrophomina phaseolina + water stressed

The rating scale for drought was from 1 to 5 according to the procedures of Auge [29], Al-Karaki et al. [30] and Olawuyi et al. Olawuyi et al. (2011b,c).

1. Excellent, normal plant growth, number of plant less than 10% water stress.

2. Good, slight drought but noticeable stunting, slight reduced leaf and number of leaf 11 -25%

3. Moderate stunting, moderate leaf reduction, number of plant 26-50%

4. Poor, heavy drought, severe stunting, reduction in leaf, leaf wilting and rolling, number of plant 51-75%

5. Too poor, very heavy drought, definite leaf with extensive and conspicuous stunting, leaf wilting, leaf rolling, severe reduction in stem diameter, severe damage and premature death.

2.7. Disease resistance of M. phaseolina by mycorrhiza fungi

2.7.1. Planting and inoculation methods

Some of the potted soil used was drenched with the Arbuscular mycorrhiza fungi (Glomus deserticola and Gigaspora gigantea) before sowing of seeds. 10 seeds of each of the pathogen susceptible cowpea varieties were sown in 2 kg sterilized sandy loam soil. Germination of seeds was observed after 4 days of planting. The plants were thinned to two seedlings per pot after 7 days of emergence. Fourteen treatments applied after 10 days of germination included; T1- cowpea (control) T2- cowpea + Glomus deserticola (drench) T3- cowpea + Glomus deserticola T4- cowpea + Gigaspora gigantea (drench) T5- cowpea + Gigaspora gigantea

T6- cowpea + Glomus deserticola + gigaspora gigantea (drench) T7- cowpea + Glomus deserticola + Gigaspora gigantea T8- cowpea + Macrophomina phaseolina T9- cowpea + Glomus deserticola+M. phaseolina (drench) T10- cowpea + Glomus deserticola + M. phaseolina T11- cowpea + Gigaspora gigantea+M. phaseolina (drench) T12- cowpea + Gigaspora gigantea + M. phaseolina T13- cowpea + Glomus deserticola + Gigaspora gigantea + M. pha-seolina (drench)

T14- cowpea + Glomus deserticola + Gigaspora gigantea + M. pha-seolina

Application of Glomus deserticola and Gigaspora gigantea were done in two forms; some pots of soil were drench with 10 g of AMF before planting, while the other treatments was applied after 10 days of plant emergence at a radius of 20 cm from the root of the cowpea plant. Simultaneously with the mycellial suspension of the pathogen (M. phaseolina) which was inoculated around wounded region of the lower stem of the seedlings after 10 days of germination.

2.8. Determination of disease incidence and severity

2.8.1. Disease incidence

The percentage disease incidence of infected cowpea seedlings was estimated as described by Persson et al. [31].

% Disease incidence = n/N x 100

Where n = number of plant showing diseased symptoms with at least a brown coloration of the stem and N= Total Number of sample used.

2.9. Disease severity

This was done using the method of Persson et al. [31] as modified by Ahmed et al. [51].

0 = Healthy plant without any visible symptoms 5 = Discolouration of less than 5 mm of the root system 10 = Discolouration of about 20 mm of the root system 25 = About 5% of the root system discoloured

50 = The whole root system discoloured but no symptom on the epicotyls or leaves

75= The whole root system as well as epicotyls discoloured with the lower leaves wilted

100 = Dead plants

2.10. Data collection and statistical analysis

Agronomic data collected at 7 days intervals include; plant height, leaf area, stem girth, number of leaves per plant, number of pod per plant and pod weight per plant were taken at the yield stage. Response of plants to drought tolerance, disease incidence and disease severity were also taken.

The leaf area was calculated using the method described by Jolaoso [32].

Leaf length x leaf width x correction factor

Where correction factor = 2.7

Statistical analysis of data were analyzed by ANOVA using SPSS version 20, while treatment means were separated using Duncan multiple range test at 5% level of probability.

3. Results

The growth response of the cowpea cultivars (IT190k-277-2 and IT84S-2246-4) to drought stress shows no significant difference on the growth parameters; plant height, leaf area and numbers of leaves at the early stage of treatment (1WAT and 2WAT), while at 3WAT and 4WAT the number of leaves of the two cultivars subjected to water stress were significantly different (p < 0.05), where treatment with Glomus deserticola (AMF) recorded higher numbers of leaves 37.33b, 36.67ab for IT190k-277-2 and 42.00b, 36.67b for IT84S-2246-4 at 3WAT and 4WAT respectively. It is also observed at the maturing stage (pod filling) 4WAT that AMF has influence on the leaf area of the cultivar IT84S-2246-4 with significant difference between the C + AMF + WS (200.48ab) and C+WS (142.17b) (Table 1).

The result of the response of cowpea cultivars to different growth stages at 7, 14, 21, 28 days before treatment and days after treatments is shown in Table 2. The observation during the before-treatment stage indicates that the growth parameters; plant height, leaf area and numbers of leaves per plant were significantly different (p < 0.05) in both cowpea cultivars IT90K-277-2 and IT84S-2446-4, except in cultivar IT90K-277-2 with the leaf area of 82.41 cm2 and 80.35 cm2 which are not significantly different (p > 0.05) at 14 and 21 days before treatment respectively. While at days after treatment a significant difference (p>0.05) is observed at 7DAT for plant height and numbers of leaves for the two cultivars.

The mean square effect of treatment, replication and growth after treatment is shown in Table 3. At before treatment stage IT90K-277-2 was not significant for plant height, leaf area and numbers of leaves per plant in both treatment and replicate levels, but highly significant (p < 0.05) for days before treatment. In IT84S-2446-4, the treatment effect was highly significant for plant height and numbers of leaves, but significant (p < 0.05) for leaf area at before treatment. The leaf area and numbers of leaves per plant were not significant at the replicate level, but highly significant for plant height. The effect of days before treatment was highly significant for all the characters. At after treatment stage, IT90K-277-2 did not produce significant effect for plant height and leaf area at both treatment and replicate levels, but significantly different for numbers of leaves per plant. There was significant effect for days after treatment in plant height and numbers of leaves, but non- significant for leaf area. Again, the height of plant

Table 1

Growth response of cowpea cultivars; 1T90K-277-2 and 1T84S-2246-4 to Glomus deserticola, Macrophomina phaseolina and their treatments.

Treatments Plant height (cm) Leaf area (cm2) No of leaves Plant height cm Leaf area (cm2) No of leaves

Initial Reading C + W 44.40a 161.61a 19.33a 36.90b 160.57a 14.00b

C + WS 38.20a 141.75a 16.00a 39.83ab 215.53a 18.00a

C + AMF + WS 40.53a 174.73a 16.00a 39.67ab 203.57a 16.00ab

C + AMF + W 40.23a 149.16a 15.00a 43.17a 219.58a 16.00ab

C + AMF + M+WS 40.40a 145.44a 15.00a 36.80b 216.11a 18.00a

1 WAT C+W 61.37a 222.39a 26.00a 49.00a 227.84a 20.67a

C + WS 51.13ab 202.75a 21.00a 53.60a 232.92a 24.00a

C + AMF + WW 50.30ab 223.24a 23.00a 57.23a 193.84a 24.00a

C + AMF + W 48.63ab 214.89a 21.33a 54.37a 252.49a 23.00a

C + AMF + M+WW 43.20b 203.07a 21.67a 50.20a 201.35a 21.67a

2 WAT C+W 88.93a 229.97a 32.00a 92.07a 233.18a 32.00a

C + WS 103.37a 210.33a 40.00a 91.77a 261.96a 29.33a

C + AMF + WS 100.90a 197.76a 35.00a 73.07a 198.52a 26.67a

C + AMF + W 88.70a 147.69a 38.00a 90.57a 225.94a 30.67a

C + AMF + M+WW 97.13a 192.69a 36.33a 72.70a 229.69a 25.33a

3 WAT C+W 117.83a 219.32a 38.67ab 115.70a 247.99a 41.00b

C + WS 103.37ab 166.45a 23.67c 83.03b 191.18a 21.67c

C + AMF + WS 85.93b 176.49a 37.33b 75.27b 228.71a 42.00b

C + AMF + W 116.03a 198.43a 49.33a 86.90ab 257.47a 51.67a

C + AMF + M+WS 105.60a 198.44a 34.67bc 74.03b 243.59a 43.67b

4 WAT C+W 118.73a 237.08a 37.33ab 120.53a 261.99a 45.67ab

C + WS 102.40ab 144.62b 14.33c 83.37b 142.17b 9.00c

C + AMF + WS 89.47b 171.95b 36.67ab 78.63b 200.48ab 36.67b

C + AMF + W 118.23a 204.64ab 51.00a 90.27b 260.55a 55.00a

C + AMF + M+WS 107.20ab 169.61b 27.00bc 75.17b 211.23ab 36.00b

Means with the same letter in the same column are not significantly different at p < 0.05 using DMRT.

Key: C- Cowpea, W- Watering, WS- Water stress, AMF- Glomus deserticola, M- Macrophomina phaseolina, WAT-week after treatment.

and numbers of leaves were highly significant at levels of treatment and days after treatment, but non-significant effect was recorded for leaf area. At replicate level, only plant height was highly significant, while leaf area and numbers of leaves per plant were not significant.

The drought tolerance of the cowpea cultivars were shown in Table 4. IT90K-277-2 shows significant difference (p <0.05) in treatment at 7DAT with C+WD (1.00) and C+AMF+WD (1.00) not significantly different. 14DAT shows significant difference in treatment with C + AMF + M+WW and C + AMF+WD not significantly different, but significantly different from C + WD and C + AMF + WW which were not significantly different, while C + WW is significantly different from the treatments. These was also the observation recorded at 21 DAT. At 28DAT C+WD, C+AMF+WW and C + AMF + M + WW were not significantly different with the value 2.00 for each treatment, but significantly

different from C + WW (5.00) and C + AMF + WD (1.00) which are significantly different.

In IT84S-2246-4, at 7DAT significant difference was recorded in the treatment with C + WD (1.00) and C+AMF+WD (1.00), C + WW (1.67) and C+AMF+WW(1.67) were not significantly different (p < 0.05) respectively. Significant difference was also observed at 14DAT with C + WD (1.33) and C + AMF + M + WW (1.67) not significantly different. 21 DAT shows non significance in treatments except C + WW (5.00) significantly different from the treatments. 28DAT recorded significant difference in the treatments except C + WD (1.33) and C + AMF + WD (1.00), C + AMF + WW (2.33) and C + AMF + M + WW (2.00) which shows non significance respectively.

The effect of treatment at different application of Glomus. deserticola and Gigaspora. gigantea on charcoal rot of cowpea is shown in Table 5. Cultivar IT90K-277-2 shows significant

Table 2

Effect of growth stages on before and after treatments on morphological characters of Cowpea cultivars T90K-277-2 and IT84S-2246-4.

Days Before treatment Days After treatment

Plant height(cm Leaf area (cm2) No of Leaves Plant height(cm) Leaf area (cm2) No of leaves

1T90K-277-2

7 DBT 16.33d 57.53c 2.00d 7 DAT 50.93c 213.27a 22.60b

14 DBT 25.31c 82.41b 8.53c 14 DAT 95.81b 195.69a 36.27a

21 DBT 30.38b 80.35b 11.20b 21 DAT 105.75a 191.83a 36.73a

28 DBT 40.75a 154.54a 16.27a 28 DAT 107.21a 185.58a 33.27a

1T84S-2246-4

7 DBT 14.20d 48.26d 2.00d 7 DAT 52.88b 221.69a 22.67c

14 DBT 21.07c 69.53c 7.93c 14 DAT 83.49a 228.68a 29.36b

21 DBT 27.15b 96.90b 10.33b 21 DAT 87.46a 236.91a 41.36a

28 DBT 39.27a 203.07a 16.40a 28 DAT 90.19a 221.20a 38.57a

Means with the same letter in the same column are not significantly different at p < 0.05 using Duncan Multiple Range Test (DMRT). Key: DBT- Days before treatment, DAT- Days after treatment.

Table 3

Mean Square effect of treatment, replicates and stages of growth on growth characters of Cowpea cultivars.

Before treatment After treatment

Source of variation df Plant Height Leaf Area No of Leaves Source of variation df Plant height Leaf area No of leaves

IT90K-277-2

corrected model 9 corrected model 9

TRT 4 17.56ns 733.88ns 5.67ns TRT 4 378.50ns 3764.32ns 367.76**

REP 2 1.90ns 1292.45ns 3.15ns REP 2 262.64ns 2094.44ns 214.72ns

DBT 3 1558.43** 26578.47** 529.31** DAT 3 10523.28** 2114.65ns 651.93**

Error 50 8.292 449.63 2.56 Error 50 167.11 1646.7 68.16

Total 60 Total 60

Corrected Total 59 Corrected Total 59

IT84S-2246-4

corrected model 9 corrected model 9

TRT 4 25.43** 1062.51* 3.92** TRT 4 1271.26** 4084.32ns 359.68**

REP 2 46.76** 324.94ns 2.47ns REP 2 836.31** 981.6ns 55.75ns

WAS 3 1698.51** 70794.75** 532.82** WAS 3 4194.61** 725.8ns 995.18**

Error 50 3.63 404.79 1.19 Error 50 207.54 1790.24 66.94

Total 60 Total 60

Corrected Total 59 Corrected Total 59

Are significantly different (p < 0.05) DMRT, (**) are significantly different (p < 0.01) DMRT, (ns) are not significantly different (p < 0.05) DMRT. Key: DAT-Days after treatment, DBT- Days before treatment, TRT-Treatment, REP- Replicate.

Table 4

Drought tolerance of two cowpea varieties as affected by Glomus deserticola and Macrophomina phaseolina.

TREATMENT 7DAT IT90K-277-2 IT84S-2246-4

14DAT 21 DAT 28DAT 7DAT 14DAT 21DAT 28DAT

C + W 1.00b 2.00b 2.00b 2.00b 1.00b 1.33bc 1.33b 1.33c

C + WS 1.67a 5.00a 5.00a 5.00a 1.67ab 5.00a 5.00a 5.00a

C + AMF + WS 2.00a 2.33b 2.00b 2.00b 1.67ab 2.00b 1.67b 2.33b

C + AMF + W 1.00b 1.00c 1.00c 1.00c 1.00b 1.00c 1.00b 1.00c

C + AMF + M + WS 2.00a 1.00c 1.33c 2.00b 2.00a 1.67bc 1.33b 2.00b

Means with the same letter in the same column are not significantly different at p < 0.05 using Duncan Multiple Range Test (DMRT). Key: C- Cowpea, W- Watering, WS- Water stress, AMF- Glomus deserticola, M- Macrophomina phaseolina, DAT- Days after treatment. Values are drought scale ratings of 1-5; l(Excellent), 2(Good), 3(Moderate), 4(Poor), 5(Too poor).

Table 5

Effect of Glomus deserticola and Gigaspora gigantea on charcoal rot by M. phaseolina.

IT90K-277-2 IT06K-123-1

Treatment Plant height Leaf area Stem girth No. of leaves Plant height Leaf area Stem girth No. of leaves (cm)

(cm) (cm) (cm) (cm) (cm) (cm) (cm)

Cowpea 40.88ef 123.59a 1.81cd 16.67e 37.20d 164.79ab 2.01a 14.25fgh

Cowpea + G. ds drench 44.12cde 134.95a 1.75cd 17.50de 39.98bcd 153.75abc 1.87ab 16.08abcde

Cowpea + G. ds simul. 35.12g 93.04de 1.69d 13.67f 38.96bcd 157.06ab 1.80bc 16.33abcde

Cowpea + G. gi drench 45.33bcd 119.74ab 1.7cd 18.17de 44.04a 171.19a 2.03a 17.50a

Cowpea + G.gi simul. 39.75f 89.34e 1.75cd 20.42bc 37.25d 127.04cd 1.90ab 15.58cdef

cowpea + M.phas. 31.13h 62.55f 1.52e 9.50g 30.05f 99.70e 1.57d 10.58i

cowpea + G.ds + M.phas + drench 44.06cde 113.00bcde 1.75cd 19.25cd 37.62cd 146.40abcd 1.79bc 13.58gh

cowpea + G.ds + M.phas + simul. 40.88ef 95.27cde 1.73cd 16.50e 34.50e 121.71de 1.71c 13.00h

Cowpea + G. gi + M.phas drench 49.48a 113.47bcd 1.93b 23.83a 39.15bcd 125.06d 1.83bc 15.00efg

Cowpea + G.gi + M.phas + simul. 42.74def 122.49ab 1.81cd 19.33cd 40.32bc 138.29bcd 1.90ab 15.67bcdef

Cowpea + G.ds + G. gi + M.phas 44.12cde 117.52bc 1.83bc 20.33bc 40.94b 143.05abcd 1.87ab 15.17def

drench

Cowpea + G.ds + G.gi + M. 42.71def 111.9bcde 1.77cd 17.42de 39.71bcd 158.86ab 1.83bc 16.75abcd

phas + simul.

Cowpea + G.ds + G. gi drench 49.11ab 143.61a 2.10a 22.25ab 41.76ab 170.06a 1.88ab 17.25ab

Cowpea + G.ds + G.gi simul. 48.08bc 119.7ab 2.07a 20.67bc 41.08b 155.62ab 1.93ab 16.83abc

Means with the same letter in the same column are not significantly different at p < 0.05 using Duncan Multiple Range Test (DMRT). Key: G. ds- Glomus deserticola, G. gi- Gigaspora gigantean, M. phas- Macrophomina phaseolina, simul.- simultaneous.

differences (p <0.05) within the treatment for the characters. Application of G. deserticola and G. gigantea were effective in ameliorating the negative effect of M. phaseolina, while soil treated with G. gigantea before planting (drench) + M. phaseolina recorded

the highest value in plant height and numbers of leaves with 49.48 cm and 23.83 respectively. It is observed in the plant height that treatment with Cowpea + G.deserticola drench, cowpea + G. deserticola+ M.phaseolina + drench and Cowpea + G.deserticola + G.

gigantean + M.phaseolina drench were not significantly different (p < 0.05). The control treatments combination of G. deserticola + G. gigantean drench (143.61cm2) and cowpea alone (123.59 cm2) recorded the highest value of leaf area with cowpea alone (123.59cm2), cowpea + G. deserticola drench (134.95cm2) and cowpea + G. deserticola + G. gigaspora drench (143.61cm2) not significantly different. Significant difference was recorded on the stem girth with cowpea + G. deserticola+ G. gigaspora drench and cowpea + G. deserticola+ G. gigaspora recording the highest value of 2.10 and 2.07 cm respectively. Also, treatment with the pathogen alone (Macrophomina phaseolina) recorded least value in all the growth parameters; plant height (31.13 cm), leave area (62.55 cm2), stem girth (1.52 cm) and numbers of leaves (9.50). Result also show that of all treatments G. gigantea and the combination of G. deserticola + G. gigantea were the most effective in the cultivar.

The cultivar, 1T06K-123-1 shows significant difference p <0.05 between the control and other treatments in all the growth characters. For plant height result shows that cowpea + G. deserticola drench (39.98 cm), cowpea + G. deserticola simul (38.96 cm)., cowpea + G. gigantean +M. phaseolina drench (39.15) and cowpea + G. deserticola+G. gigaspora+M. phaseolina simul. (39.71 cm) were not significantly different (p <0.05). There was significant difference in leaf area with cowpea + G. deserticola+M. phaseolina drench (146.40cm2) and cowpea + G. deserticola + G. gigaspora+M. phaseolina drench (143.05 cm2) not significantly different. Treatments recorded significant difference for stem girth with cowpea alone and cowpea + G. gigaspora drench recording the highest value of 2.01 and 2.03 cm respectively. There was variation in numbers of leaves, showing significant difference in treatment except cowpea + G. deserticola drench (16.08) and cowpea + G. deserticola simul. (16.33) which were not significantly different. However, the control cowpea + G. gigantea (drench) recorded the highest value in all growth parameters followed in the trend is G. deserticola + G. gigantea and cowpea alone. While treatment with cowpea + M. phaseolina recorded the least value in growth characters.

Table 6 shows that disease severity was significantly different (p < 0.05) between the treatments and control which shows no incidence of disease. 1n cultivar 1T90K-277-2 treatment with M. phaseolina was significantly different from treatment not inoculated. Treatment with cowpea + G. deserticola+M. phaseolina simul. (7.92), cowpea + G. gigantea+M. phaseolina simul. (7.92), cowpea + G. deserticola + G. gigantea drench and simultaneous (5.83) were not significantly different. Treatment with the combination G. deserticola + G. gigantea (drench and simultaneous) was most effective recording resistance. While, treatment with cowpea + G. gigantea (drench) and cowpea + G. deserticola (drench) were the

least effective in the cultivar and Cowpea + M. phaseolina recorded high susceptibility. Cultivar 1T06K-123-1 shows high susceptibility to the pathogen while treatments with AMF werw resistant.

4. Discussion

Watanabe et al. [3] reported that there are variation tolerating ability of some cowpea genotypes to drought condition in the vegetative state. On the other hand, Turk et al. [5] cited in Ahmed and Suliaman [6] also affirmed that cowpea is highly sensitive to water stress during the flowering and pod-filling stages. These were in accordance with the observations in this study as the two cowpea cultivars (1T90K-277-7 and 1T84S-2446-4) responded in variance to drought when inoculated with Glomus deserticola at vegetative stage. 1T84S-2446-4 had higher tolerance to drought compared to 1T90K-277-7. The performance of G. deserticola treated plants also agreed with the findings of Ruiz-Lanzo et al. [33], and Olawuyi et al. Olawuyi et al. (2014a,b). The increase in plant height due to period of inoculation, production of numbers of leaves and leaf area of cowpea cultivars inoculated with G. deserticola were similarly observed by Quilambo [34] and Olawuyi et al. [26]. Prolong water stress observed from the study could be due to the reduction of G. deserticola effectiveness as reported by Ryan and Ash [35] and Bryla and Duniway [36], as mycorrhiza fungi may alleviate moderate drought stress but becomes ineffective in severe drought condition.

The reduction in number of pod per plant and seed per plant were observed in treatment with water stress compared to the control. This supported the observation reported by some other researchers [37]. Although, Ravindra et al. [38], attributed the loss in seed yield to low fruiting efficiency and lack of filling time for pods, while Turk and Hall [39] linked the reduction in seed yield under water stress to the secondary detrimental effect of drought avoidance on CO2 assimilation. The significant reduction in number of harvested pods per plant under water stress could also be attributed to abscission of the reproductive structure [40].

The plant treated with G. deserticola exhibited an improved drought tolerance compared to the non mycorrhizal treatment under stress. This confirmed the report of Smith and Read (1997) who stated the ability of Vesicular Arbuscular Mycorrhiza (VAM) fungi to substantially increase the host plant's tolerance to water stress. Auge [29] reported that the mechanism used by VAM increased the root hydraulic conductivity, improved stomatal regulation, osmotic adjustment in host and enabled extraction of water from smaller pores through improved contact with soil particles as a result of the hyphae binding effect.

Table 6

Severity index and susceptibility class of cowpea cultivars 1T90K-277-2 and 1T06K-123-1 to charcoal rot disease.

Treatment Disease severity index Plant rating Susceptibility/resistance class

1T90K-277-2 1T06K-123-1 1T90K-277-2 1T06K-123-1 1T90K-277-2 1T06K-123-1

Cowpea 0.00d 0.00c 0 0 Highly resistant Highly resistant

Cowpea + G. ds drench 0.00d 0.00c 0 0 Highly resistant Highly resistant

Cowpea + G. ds simul. 0.00d 0.00c 0 0 Highly resistant Highly resistant

Cowpea + G. gi drench 0.00d 0.00c 0 0 Highly resistant Highly resistant

Cowpea + G.gi simul. 0.00d 0.00c 0 0 Highly resistant Highly resistant

cowpea+ M.phas. 42.08a 58.30a 5 4 Highly susceptible Highly susceptible

cowpea + G.ds + M.phas + drench 12.92b 7.50b 3 2 resistant resistant

cowpea + G.ds + M.phas + simul. 7.92c 8.75b 2 2 resistant resistant

Cowpea + G. gi + M.phas drench 12.5b 10.00b 3 2 resistant resistant

Cowpea + G.gi + M.phas + simul. 7.92c 9.58b 2 2 resistant resistant

Cowpea + G.ds + G. gi + M.phas drench 5.83c 5.00bc 2 1 resistant Highly resistant

Cowpea + G.ds + G.gi + M.phas + simul. 5.83c 5.00bc 2 1 resistant Highly resistant

Cowpea + G.ds + G. gi drench 0.00d 0.00c 0 0 Highly resistant Highly resistant

Cowpea + G.ds + G.gi simul. 0.00d 0.00c 0 0 Highly resistant Highly resistant

There was interaction of VAM fungi with plant pathogenic organism. The establishment of VAM in the root of host plant, primarily reduced diseases caused by soil borne pathogen [41]. Previous findings had also revealed that mycorrhizal plant offer increased tolerance to fungal root pathogen [22]. In this study the effect of the interaction between G. deserticola, G. gigantea and M. phaseolina investigated, withstand the effect of pathogen which could have resulted in great reduction in plant height and numbers of leaves which might likely reduce the photosynthetic ability of the plant thus, resulting into yield loss. Therefore plants treated with both pathogen and AMF attained normal growth. Dar et al. [42], had earlier reported that enhance plant growth improved nutrient assimilation and physical barrier that offered resistance to the plants.

The observation from this study shows soil drenched with AMF before planting improved cowpea plant compared to the un-inoculated soil before planting. This could be as a result of the potential of AMF in making the immobile phosphorous readily available in the soil. Several investigations indicate that there is a beneficial effect of VA- fungi inoculation on nutrient uptake and plant growth especially in sterilized soils [43,44]. The combination of AMF (G. deserticola and G.gigantea) were the most effective in improving the tolerance traits and disease resistance of the cowpea cultivars.

The M. phaseolina and AM fungi exploited common resources which included infection site, space and photosynthate within the root [45]. Interference competition may also arise if there is carbon availability within intercellular spaces and rhizophere [46]. The number of infection loci within the root system was reduced as a result of AM fungal colonization

[47]. The two cowpea cultivars showed high susceptibility to M. phaseolina, but, AMF inoculated plant offered resistance to the pathogen. The potentials of AMF in protecting plants from root pathogens which included; Phytophthora parasitica or Fusarium sp., root- invading nematodes had been previously reported [48,49,22,50,23].

5. Conclusion

It is evident from this study that IT 84S-2446-4 had better tolerance to drought compared to 1T90K-277-7 when inoculated with Glomus deserticola, the treatment combination of Arbuscular mycorrhiza fungi (Glomus deserticola and Gigaspora gigantea) had better potentials to tolerate the effect on susceptible Cowpea cultivars(1T90K-277-7 and 1T84S-2446-4) to drought as well as resisting the pathogen (Macrophomina phaseolina) causing charcoal rot disease of cowpea.

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