Bio-organic fertilizer plays a significant role to conserve suitable soil environment for plant growth and development. Trichoderma-enriched bio-organic fertilizer (BOF) was evaluated for bottle gourd cultivation to assess suitable dose and mode of application at field condition. The experiment was conducted in two successive years (2014/15 and 2015/16) with three replications and eight treatments of standard dose (SD) of NPK fertilizer and different doses of BOF in two modes of application. Application of BOF with 25% N of standard nitrogen dose played superior roles in present study. The highest dose (3kg/pit with 25%N+100%PK) of BOF offered the highest yield which was statistically similar to SD of NPK in both the years. But sole application (SA) of the highest dose at planting time provided 10.25% higher yield of bottle gourd compared to SD of NPK application. Conversely, 13.95 and 7.69% lower yield was monitored in first and second year, respectively by the split (double) application of the same amount of BOF compared to SD of NPK application. Trichoderma-enriched bio-organic fertilizer could save at least 75%N fertilizer usages and ensured earlier fruit setting in bottle gourd production. In the present study the highest dose (i. e. 3kg/pit of BOF & 25%N with 100%PK as of standard dose of NPK) and sole application of BOF presented superior performance to split application. Trichoderma enriched bio-organic fertilizer could serve prospective and potential roles in promotion of crop cultivation with reducing usages of N-fertilizer in future.

Keywords:bottle gourd, male and female flower, fruit diameter, fruit length, yield, sole, split application

Excessive inorganic fertilizers and plant protection chemicals usages in crop production induce risks of both human health and environment. Besides, generally 50% of N and 90% of P from applied inorganic fertilizers are fixed up in soils and run off, and ultimately it releases into atmosphere and water sources, which contribute for greenhouse gas generation, eutrophication and soil salinization.^1,2 Application of excessive P induce cadmium in soil that come to food chains.³ Moreover nitrogen application increase nitrates in ground water which are finally responsible for N-nitroso compounds and ultimately often linked with fatal methemoglobinemia.⁴ To minimize these problems the relevant researchers have been trying to develop sustainable safe and environmental friendly technologies that will help to minimize usages of chemical fertilizers for crop production. Accordingly Trichoderma spp. enriched bio-organic fertilizers; pellets and suspension are being evaluated to monitor its performance in different crop production.^5‒7 In recent years it has achieved recognition as plant growth promoter, enhanced antioxidant compounds,^7,8 increased yield of several crops in field condition.^6,10‒12 Besides, some Trichoderma rhizosphere-competent strains have been shown to have direct effects on plants, increasing their growth potential and nutrient uptake, fertilizer usage efficiency, percentage and rate of seed germination, and stimulation of plant defense against biotic and abiotic damages.^13,14 Trichoderma metabolites and roots colonization by Trichoderma change the proteome and transcriptome of plants,^15,16 which enhance plant growth and development but these mechanisms of Trichoderma plant interaction are not quite easy.

Application of Trichoderma fungi ensures the favorable environment for growing and proliferation of abundant healthy roots and simultaneously plant derived sucrose which is an important resource provided by the Trichoderma spp. Cells.¹⁷ Thus plants become benefited from this relationship through increased root and shoot growth and increased macro- and micronutrient uptake. Therefore, Trichoderma spp. are being treated as growth promoting (bio-organic fertilizer) as well as pathogen control agent (mycofungicide) and their application may lower the production cost of crops along with conserving congenial environment. Vegetable cultivation is one of the most important and dynamic wing of agriculture in Bangladesh. Among the cultivated vegetables in Bangladesh, bottle gourd (Lagenaria spp.) a cucurbit is a popular crop has now drawing attention to greater extent. Bottle gourd (Lagenaria spp.) is a common and delicious winter vegetable in Bangladesh. Generally bottle gourd fruits are used as fruit vegetable, but its leaves tender stem are used as delicious and nutritious leafy vegetable.¹⁸ It is reported as an easily digestible vegetable which keeps the body cool and prevents constipation. Its cultivation and usages are wide in winter but now a day it is cultivating during summer and rainy season also. Bottle gourd is vine crop grow rapidly and their stem become 10-12 meter long which generally need support to climb by the pole along the stem. It prefers well-drained, moist, rich soil and proper fertilizer management practices for successful crop growth and yield.¹⁹ At this situation, slow release nutrient rich fertilizers/compost/bio-organic fertilizer might contribute significant roles for bottle gourd cultivation. Bio-organic fertilizer is defined as the fertilizer contained living cells or latent cells of efficient strains of microorganisms with decompose organic substances that help crop plants in uptake of nutrients by their interactions in the rhizosphere. They help in restoring soil health and thus provide a cost effective way to manage crop yield along with balancing the environment.²⁰ The integrated nutrient management i.e. minimum usages of chemical fertilizers with organic materials such as value added bio-organic fertilizer, animal manures, crop residues, green manuring and composts are alternatives to avoid excessive usages of nitrogenous and phosphorus contained fertilizers that have enough chance to pollute our soil and environment.^1,2,5,21 Therefore, it is important to evaluate the impact and usages technology of Trichoderma-enriched bio-organic fertilizer in different crops. The present study was undertaken to evaluate the performance of Trichoderma-enriched bio-organic fertilizer in combinations with chemical fertilizers on growth, yield and yield contributing characters of bottle gourd under field condition and to assess the suitable dose and mode of application of bio-organic fertilizer in bottle gourd cultivation.

Experimental location and period

The present research project was conducted in research field of the Department of Environmental science, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur, Bangladesh in two successive years at the period of October to March in 2014-2015 and 2015-2016 years. Moreover, the experiment was conducted in same field in both the years. The experimental site is situated in the sub-tropical climatic zone. The experimental period was relatively scanty rainfall, low humidity; low temperature (Min. 12-15 and Max. 24-27°C), high light intensity and short day period prevail during this time every year in Bangladesh.

Materials used in the experiment

Bio-organic fertilizer (BOF) i.e. Trichoderma-enriched bio-organic fertilizer (Trichoderma harzianumT22) was collected from Natore Development Society (NDS) and M/S RASH Agro Enterprise, Natore, Bangladesh for conducting the present experiment. Seeds of field grown variety of bottle gourd recognized as ‘Khatlau’ were collected from the Lal teer, a prominent private agro seed business center. The fruit of bottle gourd was intermediate to long in size of dark to pale green color with light to pale colored spot that make the fruits attractive appearance.

Nutritional and biological status of soil and Trichoderma-enriched BOF

Nutritional and biological status of the soil of the experimental plot and Trichoderma-enriched bio-organic fertilizer (BOF) were analyzed in prior to fix the standard doze of chemical fertilizers for proper growth and development of the studied crop. However, the nutritional status of soil and BOF was analyzed based on procedures of Jackson,²² Nelson & Sommers,²³ Olsen & Sommers,²⁴ and microbial status of BOF was assessed based of technique of Martin⁷ & Khan et al. ²⁵ The recoded data was presented in Table 1.

Growing seedlings in poly bags

Seeds were soaked in water for overnight and then single seed was sown in each polythene (poly) bag on 15 November 2014 in first year and on 29 October 2015 in second year for rising seedling. Used polythene bags were 9cm diameter and 12cm long. Around 700g of well mixed of clay loam soil and decompose compost (1:1) was taken in each poly bag. After sowing, the poly bags were kept with care until seedling emerged and protect the seedlings from heavy drops of water, severe sunshine and red pumpkin beetle. Light irrigation was done in the late afternoon by using a fine meshed nozzle while it was required.

Experimental treatment and design

The experiment was conducted in a randomized complete block design (RCBD) with four replications and eight treatments. The unit plot size was 3.0m×2.0m and plot to plot 0.5m, row to row 0.5m distance were maintained. Single pit was allowed in each plot. The treatments were randomly assigned to different plots of each block.

However, the eight treatments were:

T₁ = Control [No inorganic fertilizer and Trichoderma-enriched bio-organic fertilizer (BOF)]

T₂ = Standard dose of NPK (130g of urea, 152g of TSP, 42g of MoP per pit). The standard dose was calculated based on fertilizer guide of Bangladesh Agricultural Research Council.²⁶

T₃ = BOF [2kg/pit, total/sole application (TA) i.e. only at planting time] + [25% N + 100% PK (based on T₂)]

T₄ = BOF (2.5kg/pit TA) + [25% N + 100% PK (based on T₂)]

T₅ = BOF (3kg/pit TA) + [25% N + 100% PK (based on T₂)]

T₆ = BOF [2kg/pit, in two equal splits application (SA i.e. two times application, planting time and 35days after transplanting] + [25% N + 100% PK (based on T₂)]

T₇ = BOF (2.5kg/pit in SA) + [25% N + 100% PK (based on T₂)]

T₈ = BOF (3 kg/pit in SA) + [25% N + 100% PK (based on T₂)].

The dose of BOF was recommended by bio-organic fertilizer company for bottle gourd cultivation.

Land and pit preparation

Well ploughed land was used for the present experiment. A single pit of size 40cm³ was prepared in each plot. The soil of the pit was prepared properly by mixing assigned treatment materials just a day before of transplanting date of the seedlings.

Application of fertilizer and Trichoderma-enriched BOF

The chemical fertilizers urea for N, triple super phosphate (TSP) for P and muriate of potash (MoP) for K were applied at the rate of 130, 152 and 42g per pit, respectively against the treatment (T₂). The dose of P and K were applied at the time of pit preparation to all treatments. In treatment T₂ urea was applied in three equal splits at pit preparation, 28 and 56 days after transplanting (DAT) of seedlings. The TA i.e. total/sole application of Trichoderma-enriched BOF was performed at pit preparation. However, in split application (SA) of BOF was carried out as 50% at pit preparation and remaining 50% was at 5weeks after transplanting of seedlings (i.e. at 35 DAT). In treatments (T₃‒T₈) the 32.5g urea or 25% of standard dose (i.e. 130g urea) was applied is three equal splits as treatment T₂.

Seedling transplanting and inter-cultural operations

Seedlings were grown in poly bag and two seedlings of 21 days old were transplanted per pit to the main field on 6 December 2014 in first year and 19 November 2015 in second year as per design of the experimental layout. The plants were always kept under careful observation, irrigation, gap filling, weeding, mulching and necessary other cultural operation were done throughout the growing season.

Assessment of microbial population density

Microbial population i.e. fungal and bacterial population density of rhizosphere soils was assayed at the middle stage (i.e. 45 DAT) of the crop bottle gourd based on procedures of Martin⁷ & Khan et al.,²⁵ respectively.

Parameter studied and harvesting fruits

Fruits of all treatments were harvested at edible maturity. Data on the following parameters were recorded to observe the impact of Trichoderma-enriched bio-organic fertilizer in combinations with chemical fertilizers on growth and yield of bottle gourd cultivation in field condition. However, the studied parameters were first flower initiation dates and total number of flowers both male and female; fruit length, diameter, weight and number; root and shoot dry weight;¹⁰ and microbial population density in rhizosphere soils.^7,25

Statistical analysis

The recorded data of various parameters were statistically analyzed using Least Significance Difference (LSD) package program. The significance of difference among the treatments of replicated means was evaluated by LSD test at 5% levels of probability. Tests were conducted using SPSS version 16.

Sex expression

Male flower - The flower initiation date is presented in DAT, which were almost similar among all treatments except T₁, where the first male flower initiation was delayed 1-2 weeks compared to other treatments in both years (Table 2). In first year it was 56 and the second year 60 DAT. While, in other treatments the male flower initiation was completed within 45-49 DAT in first year and within 47-50 DAT in second year. The flower initiation date was more or less close among the treatments and within the two years. Conversely, the variation of days within the two years in respective of treatments was quite narrow; the maximum was recorded in treatment T₁. The lowest number (i.e. 37 & 30) of male flowers was recorded in control treatment (T₁) in both years. But, the highest number was monitored in treatment T₅ that was 72 in first year and 70 in second years. These were closer to the results of T₂ and T₄ treatments. Moreover, the obtained results clearly implied that the standard dose application i.e. treatment T₂ and total/sole application (i.e. TA) of Trichoderma-enriched bio-organic fertilizer treatments (i.e. T₃, T₄ and T₅) provided the maximum number of male flowers than the others. Relatively lower number of flowers was recorded in splits application (SA) of Trichoderma-enriched bio-organic fertilizer application treatments (i.e. T₆, T₇ and T₈). Conversely the figures of required days of flower initiation and number of produced flowers in a particular treatment between two successive years were close to each other i.e. deviation was quite narrow except treatment T₁. It might be happened due to similar trend of physical factors (light intensity, temperature, humidity, moisture in soil, rainfall etc) generally prevail in similar pattern in every year at a particular location of Bangladesh. Moreover, same field and same doses of treatments were used in both years. Poor performance in control treatment (T₁, not applied any fertilizers) was noticed due to deficient nutrient there. Total/sole application of Trichoderma-enriched BOF (i.e. treatments T₃-T₅) provided similar performance on male flower production as standard dose of NPK treatment (T₂) but number of produced male flowers was quite lower in Trichoderma-enriched BOF treatments (T₆-T₈) while it was supplied in split application (Table 2). Perhaps split application of BOF provided insufficient flow of nutrients supply and favorable environment in rhizospheric regions which might induce poor production of flower. Combination of inorganic and bio-organic fertilizers helped to increase uptake of nutrients which promoted faster plant growth of plants leading to enhance reproductive development. Moreover, sufficient bio-organic fertilizer (compost) behaved as slow release of nutrients supplier and conserved favorable environment for proper root growth and development along with reducing load of chemical fertilizers usages.^5,21 The nature of flower initiation among cucurbits is not same. It varies among the members and species. But similar trend of results and findings were reported by Das et al.²⁷ & Sharma et al.,²⁸ in pointed gourd. Moreover male flower initiation in cucumber was reported in 35.25 days by Nirmala et al.²⁹

Initiation of female flowers followed similar profile as male flowers (Table 3). Like male flower, the plants of treatment T₅ produced the highest number of female flowers (i.e. 34 & 32) both in first and second year, which were closer to the results obtained in treatment of T₄. Relatively wider variation on number of female flower among treatments was observed in second year than the first year. The lowest number of female flower (23 and 20 in first & second year, respectively) was recorded in control i.e. T₁ treatment. The recorded results implied that the highest dose of BOF (i.e. 3kg/pit, treatment T₅) as total/sole application (TA) produced the highest number of flowers, but same amount of BOF in split application (treatment T₈) produced lower number of both flowers as control treatment (Table 2) (Figure 3). Spit application (SA) of Trichoderma-enriched bio-organic fertilizer treatments presented lower number of flowers compared to sole/total application. Insufficient and inconsistent nutrients supply might be happened in split application than the sole application that compelled to produce lower number of flowers. The narrow differences of flower initiation days among the treatments and between the two successive cultivated years were monitored in all treatments except T₁ (Table 3). Also similar profile (i, e. narrow deviation) was noticed for total number of flowers. The T₁ required the maximum days for first flower initiation, 62 DAT in first year and 69 DAT in second year and total number of flowers were the lowest in both years than the others. Flower initiation was happened in close distances (50-52 DAT in first year and 51-53 DAT in second year in remaining treatments. Insufficient/low nutrient supply delayed flower initiation due to insufficient/improper vegetative growth of the plant. Combination of inorganic and bio-organic/organic fertilizers enhanced to uptake different nutrients which promote faster plant growth leading to increase production of higher number of flowers. Similar profiles of findings were noticed in ridge gourd³⁰ and in muskmelon cucurbits.³¹

Fruit length and diameter

Length and diameter of bottle gourd were influenced by Trichoderma-enriched bio-organic fertilizer (BOF) application. In the first year significantly (P≤0.05) the highest length of fruit was recorded in T₃ (44.00cm) and T₅ (44.00cm) presented in Table 4. On the other hand the second highest length was recorded in three treatments T₂, T₆ and T₈. The lowest fruit length was monitored in T₁ treatment (36.00cm) followed by T₇ (37.33cm). In case of second year the highest length of fruit was found in T₅ (46.66cm) treatment which was statistically similar to the treatment of T₄. The lowest fruit length was recorded in T₁ treatment (34.66cm) followed by T₆ (36.33cm) treatment. However, the obtained records implied that superior performance was recorded in treatment T₅ in both years. As like as other parameters the fruit length was significantly influenced in treatment T₅ by Trichoderma-enriched bio-organic fertilizer application. Generally balanced and sufficient nutrients supply ensures the proper growth and reproductive development, perhaps it was ensured by the treatment of T₅. Fruit diameter was measured in neck, middle and bottom of the bottle gourd. Significantly (P≤0.05) the highest diameter in everywhere was recorded only in treatment T₅ in both years. Finally it provided the highest average diameter. However, the treatment T₃ provided statistically similar findings as T₅ in most of the cases, similarly the average diameter attained in second highest there. Conversely, the average diameters of treatments T₂, T₆, T₇ and T₈ were close to each other and the lowest record was attained in T₁ treatment. The increased fruit length, fruit cavity, fruit volume and diameter could be attributed to balanced nutrition, better nutrient uptake and synthesis of more carbohydrates by plants when provided with combinations of inorganic and bio-organic/organic fertilizers, and Trichoderma-enriched BOF which influenced on increased fruit length fruit diameter, fruit volume and fruit cavity.³² Significant increase of leaf chlorophyll content and uptake of different nutrients including micronutrients were reported in tomato by Trichoderma-enriched BOF application in field.

Fruit number and yield of bottle gourd

Fruit number and yield of bottle gourd were significantly influenced (P≤0.05) by Trichoderma-enriched bio-organic fertilizer (BOF) application. Two successive cultivated years provided almost the similar trend of results. Significantly (P≤0.05) the highest fruit number per plot was recorded in the treatment T₅ in both years i.e. 10.0 and 11.33 fruits per plot in first and second year, respectively (Table 5). However, significantly the second highest numbers of fruits were attained in treatments T₂, T₆, T₇ and T₈ in both first and second years. Furthermore earlier fruit setting and increased number fruits were noticed in promoted BOF treatment compared to standard dose of NPK application (Figure 1). Within these four treatments statistically similar number of fruits was monitored in respective years. The lowest number of fruits was recorded in treatment T₁, which was statistically similar to the treatments of T₃ and T₄ in both the years. Moreover in both the years statistically similar and highest bottle gourd yield (kg/plot) was attained in three treatments T₂, T₅ and T₈ i.e. standard dose of NPK fertilizer, 3kg/pit (the highest amount) of BOF as sole application (one time) and 3kg/pit of BOF in split (two times) application treatments, respectively. But the second highest yield was attained in treatment T₆ in both the years and the lowest yield of bottle gourd was recorded in control (i.e. T₁) treatment. The fruit yield depends on fruit number per plot, fruit length and fruit diameter. The fruit length and diameter were recorded significantly higher in all cases of two successive years at T₅ treatment (Table 4). The average fruit length and diameter of T₃ was second highest and closer to value of the treatment T₅. But the yield (kg/plot) of bottle gourd in treatment T₃ was significantly poor not similar as T₂, T₅ and T₈ treatments because of average lower number of fruits per plot in both years (Table 5) as well as higher number of deformed fruits. Deformed fruits were observed in almost all treatments but it was maximum in treatment T₃ (not included). Perhaps insufficient nutrients enhanced to produce deformed fruits.

Figure 1 Promotion of bottle gourd production using Trichoderma-enriched bio-organic fertilizer application in field [T₂ – standard dose of NPK fertilizer and T₅ - 3kg/pit sole/total application + (25% N of T₂ + 100% PK of T₂)].

Maximum percent yield increase over standard dose of NPK (i.e. T₂ treatment) was recorded in treatment T₅ i.e. Trichoderma-enriched bio-organic fertilizer application. Only treatment T₅ provided 10.25% higher fruit yield over standard dose of NPK application. Conversely the fruit yield of bottle gourd 7.69, 10.30, 25.66 and 65.93% lower in treatments T₈, T₆, T₃ and T₁, respectively compared to T₂ i.e. standard dose of NPK fertilizer application. The findings of the present research project clearly presented that the Trichoderma-enriched bio-organic fertilizer 3kg/pit as sole (one time) application provided the 10.25% higher yield compared to the standard dose of NPK application. But the achieved yield at treatment T₅ was statistically similar to the yields of T₂ and T₈ treatments. Amount of inorganic and Trichoderma-enriched bio-organic fertilizer was same T₅ and T₈ treatments but application mode of BOF was different. However, the treatments T₅ and T₈ were consisted 75% less N compared to T₂ treatment; it means that practicing of T₅ and T₈ treatments would help to save 75% of N usages in bottle gourd cultivation. Above 50% of N fertilizer usages was supplemented by Trichoderma-enriched compost application in maize,⁵ in mustard⁶ and in tomato yield and quality¹⁰ in field condition were reported. Increased yield of several crops such as in rice,⁹ in wheat¹¹ and in soybean¹² were reported by Trichoderma spp application. Application of Trichoderma spp and enriched bio-organic fertilizer augmented to solubilize of different nutrient elements and enhanced harboring of microbes in rhizosphere soils.⁷ Moreover, Vinale et al.³³ reported that the application of Trichoderma spp dramatically increased the number of fruits per plant in pepper and tomato grown in greenhouse than untreated control. It has been reported that the strains of different Trichoderma species showed variable responses in cucumber, loofah and bitter gourd.³⁴ Increase yield of wheat was found similar to the findings of Sallam et al.³⁵ where the formulation of Trichoderma spp treatments enhanced green yield of bean plants compared to control. The increase in yield can also be attributed to the application of Trichoderma harzianum (Th3) bioformulation along with the farm yard manure helped in increasing the colonies by providing nutrient to Trichoderma thereby increasing the plant growth and yield of wheat. Generally 20% decrease in yield is expected in plants cultivated in organic systems than conventionally produced crops.³ But in the present experiment would not conducted with sole application of bioorganic fertilizer, therefore percent yield losses only in Trichoderma-enriched bio-organic fertilizer application was not computed.

Dry matter production

Impact of Trichoderma-enriched bio-organic fertilizer application on dry matter production of bottle gourd was presented in Table 6. The highest shoot dry weight (1102.70 and 1201.70gplot^-1 in first and second year, respectively) of bottle gourd was recorded in T₅ treatment in both years followed by T₂ (1024.70gplot^-1) treatment. On the other hand the lowest shoot dry weight was recorded in T₁ in both years. Like shoot weight, the highest root dry weight (15.33gplot^-1) was noticed in T₅ treatment in first year and T₆ (19gplot^-1) in second year. But statistically similar root dry weight of T₆ was observed in T₅ treatment in second year. The lowest root dry weight was found in T₂ and T₄ in first year, and T₃ in second year. Conversely, both first and second year the highest total dry matter 1118.03 and 1220.36gplot^-1 of bottle gourd was recorded in T₅ treatment, respectively. The second highest was recorded in T₂ of first year and second year in T₂ and T₈ treatments. The lowest record was achieved in T₁ in both year also. Like other parameters the root and shoot weight was influenced significantly and attained maximum at T₅ treatment. Similar amount of Trichoderma-enriched bio-organic fertilizer but different application mode in treatment T₈ provided second height total dry weight as T₂ treatment except first year. Perhaps these treatments provided sufficient available nutrients as well as favorable rhizosphere environment in spite of biotic and abiotic stresses by increasing microbial populations.^13,14 Trichoderma promoted growth of primary root length and root branching in maize and beans by inducing lateral root growth. In plants auxins have been demonstrated to initiate lateral root growth³⁶ and the observed effects of Trichoderma in promoting lateral root development is similar to in vitro experiments performed by Hexon et al.,³⁷ that showed that Trichoderma spp. produced indole-3-acetic acid (IAA) that promoted lateral root formation in Arabidopsis thaliana. Increased root size resulted into increased shoot size which translocate into increased shoot biomass production indicating a beneficial effect of Trichoderma inoculation on plant growth and development. The positive influence of Trichoderma on root system architecture would therefore relate to increased yield of plants. Trichoderma enhanced root biomass production and increased root hair development has also been reported by Björkman et al.³⁸ Similar trend of results was noticed in corn shoot dry weight using T. harzianum.³⁹

Microbial population in rhizosphere soil

Microbial population i.e. fungal and bacterial population in rhizosphere soils of bottle gourd was assayed in middle stage of the crop at 45 DAT in one year (second) only. The population density of these two groups of microbes was influenced by quantity and mode of application of Trichoderma-enriched bio-organic fertilizer (BOF) in the present experiment. The highest figure of fungal population was recorded in treatment T₈ but which was statistically similar to all Trichodema-enriched bio-organic fertilizer added treatments except T₄ (Table 7). Significantly (P≤0.05) the lowest number of fungal population was monitored in T₁ i.e. control treatment. At treatment T₂ (i.e. standard dose of NPK fertilizer application) significantly higher number of fungal population than the T_1, but lower number than T₅, T₆ and T₈ treatments. For bacterial population, the highest number was monitored in treatment T₆, which was statistically alike to T₅, T₇ and T₈ treatments. Statistically the second highest bacteria population in rhizosphere soil was noticed T₃ and T₄ treatments. The lowest number of bacterial population was recorded in treatment T₁, which was statistically similar to T₂ treatment. In the present experiment, the dose of Trichoderma-enriched bio-organic fertilizer along with inorganic fertilizers in T₃ and T₆ was same i.e. 2kg/pit of BOF, similarly in T₄ and T₇ 2.5kg/pit of BOF, and in T₅ and T₈ 3kg/pit of BOF. Only mode of application of BOF was different. In treatments T₃, T₄ and T₅, the total amount of BOF was applied at planting time. Conversely, in treatments T₆, T₇ and T₈ the total amount of BOF was applied in equal two installments at planting time and at 35 DAT. The present obtained results implied that the higher amount of BOF i.e. treatments T₅ and T₈ maintained statistically highest number of microbial populations (both fungi and bacteria) in rhizosphere soils by irrespective of application mode (Table 7). Moreover the split application of BOF maintained increased microbial population (both fungi and bacteria) in rhizosphere soil compared to sole/total application. Perhaps split application helped to maintained population trend in sustainable way but while sole application of BOF was done, then intensive competition for food and spaces among the population might be happened that resulted declined trend of population density. Besides microbial population was assessed in rhizosphere soil that was collected at 45 DAT, just after second instalment of BOF application was done at 35 DAT. It might influence the microbial population in split application treatments. Microbial population in rhizosphere soils was assayed by several authors as usages of organics improved the productivity of crops as well as microbial population soil.⁴⁰ The enhanced microbial populations and their activities in rhizosphere soils increased root growth and uptake of higher amount of nutrients by excreting organic acids and phosphates could release elements from complexes presently in soil and increase nutrient availability to plants.^41,42

Excessive usage of expensive chemical fertilizer could be avoided to a significant extent without compromising the yields by employing the Trichoderma-enriched bio-organic fertilizer. Combined application of Trichoderma-enriched bio-organic fertilizer with reduced rate of N fertilizer enhanced vegetative, reproductive growth and yield of bottle gourd by slow and steady release of nutrients to the plants than the sole application of NPK fertilizer. Trichoderma-enriched bio-organic fertilizer application enhanced earlier fruit setting and could save at least 75% N i.e., decrease cost of cultivation by minimizing N fertilizer usages that would reduce toxicity in food and environment.

None.

Authors declare that there is no conflict of interest.

1. Simpson RJ, Oberson A, Culvenor RA, et al. Strategies and agronomic interventions to improve the phosphorus-use efficiency of farming systems. Plant and Soil. 2011;349(1-2):89‒120.
2. Costa da PB, Beneduzi A, Souza de R, et al. The effects of different fertilization conditions on bacterial plant growth promoting traits: guidelines for directed bacterial prospection and testing. Plant and Soil. 2013;368(1-2):267‒280.
3. Rembiałkowska E. Quality of plant products from organic agriculture. Journal of Science Food Agriculture. 2007;87(15):2757‒2762.
4. Chan TYK.Vegetable-borne nitrate and nitrite and the risk of methaemoglobinaemia. Toxico Let. 2011;200(1-2):107‒108.
5. Molla AH, Fakhrul Razi A, Hanafi MM, et al. Compost produced by solid state bioconversion of biosolids: a potential source for plant growth and environmental friendly disposal. Communications in Soil Science and Plant Analysis. 2005;36(11-12):1435‒1447.
6. Haque MM, Haque MA, Ilias GNM, et al. Trichoderma-enriched biofertilizer: A prospective substitute of inorganic fertilizer for mustard (Brassica campestris) production. The Agriculturists. 2010;8(2):66‒73.
7. Khan MY, Haque MM, Molla AH, et al. Antioxidant compounds and minerals in tomatoes by Trichoderma-enriched biofertilizer and their relationship with the soil environments. Journal of Integrative Agriculture. 2017;16(3):691‒703.
8. Hermosa R, Viterbo A, Chet I, et al. Plant-beneficial effects of Trichoderma and of its genes. Microbiology. 2012;158(1):17‒25.
9. Banayo MNP, Pompe CCS, Aguilar EA, et al. Evaluation of biofertilizers in irrigated rice: Effects on grain yield at different fertilizer rates. Agriculture. 2012;2(1):73‒86.
10. Molla AH, Haque MM, Haque MA, et al. Trichoderma-enriched biofertilizer enhances production and nutritional quality of tomato (Lycopersicon esculentum Mill.) and minimizes NPK fertilizer use. Agriculture Research. 2012;1(3):265‒272.
11. Sharma P, Patel AN, Saini MK, et al. Field demonstration of Trichoderma harzianum as a plant growth promoter in wheat (Triticumaestivum L). Journal of Agricultural Science. 2012.4(8):65‒73.
12. Abudulai M, Jerry A, Nboyine JA. The effects of the microbial amendments, Eco-T (Trichoderma harzianum) and Eco-Rhizsoy (Bradyrhizobium japonica strain WB74) on growth and yield of soybean in Ghana. Advanced Crop Science. 2014;4:57‒62.
13. Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol. 2010;48:21‒43.
14. Mukherjee S. The Emperor of All Maladies: A Biography of Cancer. Pulitzer Prize Winner. 2011.
15. Marra R, Ambrosino P, Carbone V, et al. Study of the three way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Curr Gen. 2006;50(5):307‒321.
16. Alfano G, Ivey MLL, Cakir C, et al. Systemic modulation of gene expression in tomato by Trichoderma hamatum382. Phytopathology. 2007;97(4):429‒437.
17. Cai F, Yu G, Wang P, et al. Harzianolide, a novel plant growth regulator and systemic resistance elicitor from Trichoderma harzianum. Plant Physiol Biochem. 2013;73:106‒113.
18. Gupta D, Verma AK. Population fluctuations of the grubs of red pumpkin beetle; Aulacophora foveicollis (Lucas) infesting cucurbitaccous crops. Advances in Plant Science. 1992;5:518‒523.
19. Barua S. Role of Trichoderma-enriched bio-organic fertilizer for bottle gourd production. MS thesis Dept Environ Sci. 2015.
20. Vessey JK. Plant growth promoting rhizobacteria as bio-fertilizers. Plant and Soil. 2003;255(2):571‒586.
21. Meunchang S, Panichsakpatana S, Weaver RW. Tomato growth in soil amended with sugar mill by products compost. Plant and Soil. 2006;280(1-2):171‒176.
22. Jackson M L. Soil chemical analysis. Prentice Hall of India Pvt. Ltd, New Delhi. 1973;498.
23. Nelson DW, Sommers LE. Total carbon, organic carbon and organic matter. In: page AL, Miller RH, Keeney DR, editors. Methods of soil analysis Part 2 Chemical and Microbiological Properties. Am Soc Agron Inc Madison WI: USA;1982:539‒579.
24. Olsen SR, Sommers LE. Phosphorus. In: page AL, Miller RH, Keeney DR, editors. Methods of soil analysis, Part 2, Chemical and Microbiological Properties. Am Soc Agron Inc Madison WI: USA;1982:403‒430.
25. Martin JP. Use of acid, Rose Bengal and Streptomycin in the plate for estimating soil fungi. Soil Science. 1950;69(3):215‒232.
26. BARC (Bangladesh Agricultural Research Council). Fertilizer recommendation guide. Bangladesh Agricultural Research Council. 2012:260.
27. Das MK, Maity TK, Som MG. Growth and yield of pointed gourd as influenced by nitrogen and phosphorus fertilization. Vegetable Science. 1987;14(1):8‒26.
28. Sharma SK, Mehta BS, Rastogi KB. Effect of planting dates and nitrogen levels on yield and quality attributes of cucumber. Indian Journal of Horticulture. 1997;54(2):160‒162.
29. Nirmala R, Vadivel E, Azakiamanavalan RS. Influence of organic manures on fruit characters and yield of cucumber Cv. Local. South Indian Horticulture. 1999;47(1/6):65‒68.
30. Arora SK, Youdhavir S, Pandita ML. Studies on nitrogen fertilization on plant density and ethopon application in ridge gourd. Haryana Journal of Horticulture Science. 1994;23(4):313‒317.
31. Singh DN, Mishra RS, Parlda AK. Effect of nitrogen, phosphorus, potassium and spacing on flowering, fruiting and fruit characters of muskmelon. Orissa Journal Horticulture. 1995;23(1-2):70‒76.
32. Patil SD, Keskar BG, Lawande KE. Effect of varying levels of N, P and K on growth and yield of cucumber (Cucumis sativus L.) Cv. Himangi. Journal of Soils Crops. 1995;8(10):11‒15.
33. Vinale F, Sivasithamparam K, Ghisalberti EL, et al. Trichoderma plant pathogen interactions. Soil Biology and Biochemistry. 2008;40(1):1‒10.
34. Lo CT, Lin CY. Screening strains of Trichoderma spp. for plant growth enhancement in Taiwan. Plant Pathology Bulletin. 2002;11:215‒220.
35. Sallam NMA, Abo Elyousrand KAM, Hassan MAE. Evaluation of Trichoderma Species as Biocontrol Agents for Damping-Off and Wilt Diseases of Phaseolus vulgaris L. and Efficacy of Suggested Formula. Egyptian Journal of Phytopathology. 2008;36(1-2):81‒93.
36. Casimiro I, Marchant A, Bhalerao RP, et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell. 2001;13(4):84‒852.
37. Hexon AC, Lourdes MR, Carlos CP, et al. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiol. 2009;149(3):1579‒1592.
38. Björkman T, Blanchard LM, Harman GE. Growth enhancement of shrunken-2 sweet corn when colonized with Trichoderma harzianum 1295-22; effect of environmental stress. JASHS. 1998;123(1):35‒40.
39. Björkman T, Price HC, Harman GE, et al. Improved performance of shrunken 2 sweet corn using Trichoderma harzianum as a bioprotectant. Hort Science. 1994;29(5):471.
40. Ravichandra M. Effect of integrated nutrient management practices on soil microbial population in a soybean-wheat cropping sequence. Annual Agricultural Research. 1995;16(2):95‒200.
41. Rudresh DL, Shivaprakash MK, Prasad RD. Effect of combined application of rhizobium, phosphate solubilizing bacterium and Trichoderma spp. on growth, nutrient uptake and yield of chickpea (Cicer aritenium L.). Applied Soil Ecology. 2005;28(2):139‒146.
42. Cai ZC, Ma J, Li XL, et al. Effects of nitrogen fertilizer and wheat straw application on CH4 and NO emissions from a paddy rice fields. Australian Journal Soil Research. 2007;45(5):359‒367.