Plants extracts and a mycoinsecticide (Metarhizium anisopliae) in cowpea yield improvement in Guinean Savanah and Sahe- lian Savanah agro-ecological zones of Cameroun

Cowpea yield improvement is done by adding agricultural inputs. The use of natural substances as pesticides is being encouraged to fight against cowpea field pests. The pesticidal potentials of aqueous extracts of Azadirachta indica and Boswellia dalzielii, Metarhizium anisopliae, alone and in combination with plant extracts, as well as the commercial insecticide Decis were tested on two Vigna unguiculata varieties in field in two agroecological zones (Guinean Savanah and Sahelian Savanah) of Cameroon. The field trials were arranged in a completely randomized block design with nine treatments including control. Each treatment was replicated four times. Vigna unguiculata plants were sprayed at flowering stage thrice with insecticidal products at 5 days interval. Data assessment consisted of counting ramifications per plant, the number of pods per block, and seed yield. All the tested insecticides significantly (p<0.0001) improved the cowpea yield in the two agro-ecological zones. The productions parameters were highly influenced by variety and agroecological zone. The extracts and their combinations were as effective as synthetic pesticide (Decis). Bafia variety recorded the highest ramification rate (37.03±1.59) when treated with the combination of M. anisopliae and A. indica in Maroua (Sahelian Savanah). The same variety also produced most important pods number (90.50±16.66) in Ngaoundere (Guinean Savanah) with the combination of A. indica and B. dalzielii. The highest seed yield (44.23±2.31) was recorded in Ngaoundere with B125 variety treated with the combination of the three treatments (A. indica, B. dalzielii, M. anisopliae). A. indica, B. dalzielii, M. anisopliae and their combinations could be considered as potential natural input in the improvement of V. Unguiculata yield. This would not only increase V. unguiculata yield but also preserves environment from the pollution due to the use of synthetic residual chemicals.


Introduction
Cowpea (Vigna unguiculata L. Walp) is a staple food in Cameroon. It is cultivated and consumed across the ten regions of the country and the neighboring countries. The country's cowpea production increased from 40 000 tons in 1997 to 198 201 tons in 2017 [1]. Whereas the production is increasing, cowpea grain yield in farmer's fields is decreasing. Both the cowpea grain and leaves are edible products that are rich and cheap sources of high-quality protein (25-32%) and vitamins [2]. Immature pods and peas are used as vegetables while several snacks and main dishes are prepared from the grains. The seed is valued as a nutritional supplement to cereals [3]. The freshly harvested leaves are sold in local markets. Cowpea shoots and leaves are rich sources of calcium, phosphorous and vitamin B [4]. The young leaves are especially important in drought-prone regions of Sub-Sahara Africa (SSA) to tide local populations over during the "hungry period" [5]. Cowpea provides farmers with needed cash income as it is one of the first agricultural products to reach the market each year [6]. The leaves and stems are also an important source of high-quality hay for livestock feeding and roots fix atmospheric nitrogen through symbiosis with nodule bacteria [7].
The growth and production of cowpea is seriously hampered by numerous biotic factors including insect pests. Yield losses range from 10 to 100% due to the activities of wide range of insect pests which attack cowpea crop in the field at different growth stages [8]. Cowpea pests colonize the plant from seedling to the developing pods. The pest sucks leaf sap and deposits honeydew on the leaves. This sucking activity results in nutrient drain which causes direct reduction of plant productivity, the number pods and seeds size are reduced [9]. The pest is considered also as important vector of virus on cowpea [10]. Currently, exploration of bio-pesticides is gaining momentum by the agricultural industries in formulating some novel bio-agents for the management of crop pests.
Extracts of many plants could be used as an alternative to synthetic insecticides [11]. Azadirachta indica has been revealed as an insecticidal plant [12,13], it is the same case for Boswellia dalzielii [14]. However, the use of the enthomopathogenic fungus Metarhizium anisopliae has shown the insecticidal properties [15,16,17]. These properties confer to these natural products the potentialities to be used as alternative to synthetic pesticides. In order to increase effectiveness of these substances, their different combinations can be explored since the combination of B. dalzielii and A. indica extracts to the entomopathogenic fungus M. anisopliae has not yet been tested on cowpea production parameters. The efficacy of pesticides can be influenced by crop variety and agroecological zone, then the determination of their effect reveals interesting in the framework of integrated pest control. Therefore, the effect of natural substances, A. indica, B. dalzielii and M. anisopliae and their combinations on cowpea production was investigated.

Materials and Methods
The experiment was carried out in the Guinean Savannah (Dang-Ngaoundéré) and the Sahelian Savanah (Béguélé-Maroua) agro-ecological zones of Cameroon. Trials were conducted during two consecutive years (2014 and 2015), and the field working dates are summarized in Table 1. Plant material consisted of two cowpea seed varieties: the local Bafia cultivated locally during subsequent work, and the B125 provided by the Institute for Agricultural Research and Development (IRAD) of Maroua-Cameroon. The B125 variety was an early maturity variety (75 days), whereas the Bafia variety was an intermediate variety (85 to 95 days).

Figure 1. Variation of number of ramifications by agro-ecological zones, varieties and years
In Ngaoundéré (table 2), insecticide treatments significantly (p<0.0002) improved the ramifications production on Bafia variety which were not the same in B125 variety (p=0.1405) in 2014. Compared to negative control, A. indica + B. dalzielii were the bio-insecticidal treatments which considerably improved ramifications production as far as the synthetic insecticide taken as the positive control. The production of ramifications was lesser in A. indica, M. anisopliae and M. anisopliae + A. indica treatments than negative control. In 2015, all different insecticidal treatments significantly improved (p=0.0240) ramification production of B125 variety. Among them, M. anisopliae + B. dalzielii were the treatments which induced the highest ramification production. Except synthetic insecticide Decis and binary combination A. indica + B. dalzielii, all the other insecticidal treatments had improved the production of ramifications more than negative control. The different treatments significantly affected Bafia variety ramification in 2014 (p=0.0002) but in 2015 it was not the case (p=0.5288).
In Maroua in 2014, there were no significant difference of ramification production between insecticidal treatments and control (p=0.1165) on B125 variety. It was contrary on Bafia variety (p=0.0021). With 25% of more ramifications produced, A. indica treatment was the most effective. This treatment induced much ramification production than Décis. In 2015 (table 2), insecticidal treatments significantly induced more ramification production than control on the two cowpea varieties, B125 (p<0.0001); Bafia (p<0.0001). The 20% of more ramification production induced by A. indica and B. dalzielii treatment, these two treatments were more effective than Decis. The other bio insecticidal treatments during the two years of experiment in the same way induced higher ramification compared to the reference pesticide Decis. In our Savannah Sahelian zone within the two years experiment (2014 and 2015), the ternary combination M. anisopliae + A. indica + B. dalzielii was very even most effective treatment especially with B125 variety in the second year (year 2015).

Influence of insecticide formulation on pods production
In general, the pods productions varied according to sites, varieties and years (Figure 2). This pod production was higher in Ngaoundere than Maroua (t=5.58; p<0.0001). Even the pods production of B125 was higher than that of Bafia, this difference was not significant (t=0.88; p=0.3794). The production was higher in 2014 than 2015 (t=2.12; p=0.0350).

Figure 2. Variation of number of pods by Agro-ecological zones, varieties and years
Different treatments differently affected the pods productions in Ngaoundere and Maroua, and in the same zone this production varied according to the varieties (table 3). Concerning Ngaoundere, results show there is not significant different (p=0.8554) amongst the treatments on pod production of B125 variety in 2014. It was not the same case concerning Bafia variety (p=0.0492), where used bio-pesticides differently improved pod productions, that production was higher than negative control. On this variety, plots

Influence of pesticide formulations on yield
The yield of cowpea crop treated with the different formulations of bioproducts significantly varied according to the sites (t=-6.34; p<0.0001), varieties (t=13.45; p<0.0001) and years (t=3.11; p=0.0021) (Figure 3). The yield was higher in Ngaoundere than Maroua and B125 variety recorded higher yield compared to Bafia variety. Concerning the two years of cropping, the first year (2014) recorded higher yield compared to the second year (2015).
The bands with same letter on top for the same factor are not different according Student-Newman-Keuls test (P<0.05).

Figure 3. Variation of yield by Agro-ecological zones, varieties and years
At Ngaoundere in 2014, all the insecticidal formulations significantly improved yield compared to negative control on the two varieties (p<0.0001 for B125; p=0.0012 for Bafia). In B125 variety, the bioformulations induced more than 100% yield increase compared to the negative, it was the same performance reached by synthetic insecticid Decis. On Bafia variety, the single products A. indica, B. dalzielii and M. anisopliae consid- Yield per bloc Yield erably induced yield improvement compared to the negative control; this yield improvement was 25%. However, those yield improvements were lower to those induced by the combined products. The combined bio-formulations improved yield as more as Decis with an increasing of 75%. In 2015, no significantly improving of yield on Bafia variety (p=0.4110) was observed. On B125 variety, all pesticides formulations improved the yield and this was varied significantly (p<0.0001). Despite 100% of yield increasing, A. indica and M. anisopliae were the lesser bio-formulations. M. anisopliae + A. indica + B. dalzielii was the most effective bio-formulation with 200% yield increasing compared to the negative control. The influence of the different treatments in Sahelian Savannah (Maroua) significantly showed more yield improvement compared to the control (p<0.0001) on B125 variety in 2014. All biopesticides improved yield by more than 100%, what was higher than the improvement yield induced by the reference pesticide Decis. There was yield increase of Bafia variety treated with all used pesticides, and no significant difference was observed for the different formulations (p=0.9293). In 2015, all the treatments induced yield increase compared to the negative control. For a given variety the different pesticidal formulations had statistically the same performance (B125 variety : p=0.0706 and Bafia variety: p=0.6328).

Discussion
The formulated plant-based products in this study have influenced the different investigated production parameters. There were more ramifications in Maroua than Ngaoundere in the two years (2014 and 2015). The most rainfall in Ngaoundere leaches the soil, that weakens plants growth, and the Bafia variety has tendency to crawl in Maroua to resist at the drought [20]. The absence of significant difference between treatments in Ngaoundéré on B125 variety in 2014, on Bafia variety in 2015, and on B125 variety in Maroua in 2015, could be due to the late application (after vegetable growth) of different formulated pesticides. This is similar with Sharah and Ali [21] observations. Difference observed between different treatments in Ngaoundere on Bafia variety in 2014, and on B125 variety in 2015, were the result of the growing plant which is not always uniform.
Concerning production of pod, the higher production observed in Ngaoundere than Maroua was due to the variation of growing plant in different agro-eologial zone [22]. In Ngaoundere, the well full-blowing of Bafia variety in 2014 has induced a better production of pods than B125 variety. But in 2015, the soil leaching weakened Bafia variety growth and reduced the pod production compared to B125 variety. In Maroua, the Bafia variety with his intermediate cycle has been interrupted by the shoddiness of the rainfall season, then it did not produce more pods. The combination properties effects of in A. indica + B. dalzielii treatment in 2014 on Bafia variety in Ngaoundere, allowed to this treatment to produce much pods than Decis. The presence of kerosine in M. anisopliae treatment weaken plants' development, so treatments which were combined with M. anispliae produced less pods, but much than A. indica treatments which, according to Bambara and Tiemtore [23], is more effective in stocking than yield. The most resource in gum of B. dalzielii [24] makes it viscous, favourable to his adhesion that would allow B. dalzielii treatment to be more effective than A. indica treatment. According to Ibrahim [25], the adhesion is an important factor of the effectiveness of a treatment.
As regard cowpea yield, it was higher in Ngaoundere than Maroua. That shows the influence of the agro-ecological zone on crop production [22]. Then the cowpea yield in this study was affected by soil, climate and environmental conditions which characterised the different zones. The rainfall season in Maroua is shorter than the one of Ngaoundere, the intermediate cycle of Bafia variety has no more full bloom there. In Ngaoundere in 2014, with good meteorology conditions, B125 variety produced as well as Bafia variety. On B125 variety, the proprieties of different biopesticides have permitted them to improve yield as well as synthetic pesticide Decis. The lower efficiency of yield improving with A. indica and B. dalzielii extracts on Bafia variety compared to the other insecticidal treatments, were due to the fact that biopesticides have lower efficiency than synthetic pesticides as show by Dereval et al. [26]. The authors observed that the low remanence of biopesticides and the fact that their effectiveness depend to environment conditions make them less effective than synthetic chemical pesticides. These results are similar as Bambara and Tiemtoré [23] who show that A. indica, Euphorbia balsamifera and Hyptis spicigera were less effective than Decis. The presence of kerozene which have propensity to burn with high temperature was the cause of the helpless of M. anisopliae treatment. Treatments in combination, by the synergy of their different constituents, induced improving yield as well as Decis. This is similar to Barry et al. [17] who showed that the combination of M. anisopliae with other extracts improved the pesticidal efficacy than alone. In 2015, the low production of Bafia variety was due to the high rainfull which leached the soil. The synthetic pesticide Decis induced more yield improving than biopesticides in B125 variety [23,26]. Moreover, this pesticide has large spectrum and it is systemic. Among biopesticides, the higher yield observed in combination of M. anispliae + A. indica + B. dalzielii could be caused by the synergic activities of their different constituents [16,17]. The lower yield observed in A. indica treatments was due to the fact that, the efficacy of A. indica products was reduced in field [23]. This could be to the fact that the field is not a close space ass stores which increase the evaluability of active components content in pesticide. Also the field environment condition, are very fluctuating which greatly affected the efficacy of pesticide compared to storage. In Maroua, plants have benefited from properties of treatment as A. indica and B. dalzielii extracts, that permited them to produce too much than the other pesticides treatments on B125 variety in 2014. The low rainfull of Maroua reduces the leaching of these biopesticides. But when the rainfall is very weak, the need of plant of water is not well satisfied which reduced the crop production as observed in the present study on the two cowpea varieties in year 2015.

Conclusion
The aqueous extracts of A. indica and B. dalzielii, the mycoinsecticide M. anisopliae, and their various combinations have resulted in improving the cowpea full-blow. These bio-insecticides also contributed to the improving of his yield, and could therefore be proposed to substitute the commonly used synthetic pesticides in field in Guinean savannah and in Sahelian savannah. However, intermediate cycle plants as Bafia variety would be recommended to Guinean savannah than Sahilian savannah, and arrangement ought to be taken into consideration to reduce soil leaching in Guinean savannah characterised by heay rainfall.