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Approving the biocontrol method of potato wilt caused by Ralstonia solanacearum (Smith) using Enterobacter cloacae PS14 and Trichoderma asperellum T34

This study aimed to evaluate the efficiency of Enterobacter cloacae PS14 and Trichoderma asperellum T34 in the control of potato wilt, caused by Ralstonia solanacearum (Smith), under greenhouse and field conditions. In vitro, the endophyte E. cloacae PS14 caused the highest reduction of the pathogen growth among 7 screened bacteria. It produced an inhibition zone as 16.9 mm compared to a specific antibiotic (20.0 mm). E. cloacae PS14 was selected as an effective antagonistic bacterium to be compared to T. asperellum strain T34 for reduction of the disease as well as increasing the crop yield of potato plants. Both E. cloacae and T. asperellum reduced the disease severity up to 10.7–26.5%, respectively, under greenhouse and up to 26.6–36.6%, respectively, under field conditions. The results approved that both E. cloacae and T. asperellum increased the yield of the crop by 20.44– 40.96%, respectively. Their mode of action was indicated by suppression of the pathogen as well as induction of plant systemic resistance. The induction of systemic resistance was confirmed by increasing the total phenol and salicylic acid contents as well as increasing the activities of peroxidase, lipoxygenase, and polyphenol oxidase in potato plants than the healthy or only infected plants. Production of siderophore, indole-3-acetic acid (0.577– 0.884 μM), hydrogen cyanide (2.34–3.61 μg/ml), and salicylic acid (0.436–1.488 μg/ml) was confirmed by E. cloacae PS14 and T. asperellum T34, respectively, in vitro. The study recommends the new strain E. cloacae PS14, as new endophytic effective bacteria, in the control of R. solanacearum causing the potato wilt disease. Keyword: Potato wilt, Endophyte, Biological control, Enterobacter cloacae, Trichoderma asperellum, Systemic resistance Background Bacterial wilt disease caused by Ralstonia solanacearum (Smith) is one of the serious plant diseases worldwide (Peeters et al. 2013). In Egypt, as well as in many countries, it has become a severe problem for the production of potato plant (Abo-Elyousr and Bagy 2018). Traditional control methods such as short rotation, resistant cultivars, and soil fumigation have been suggested. However, such methods are not always enough effective since R. solanacearum can remain with the infested plant debris in the soil for a long time. Recently, the research focuses on some alternative methods to control the disease that are characterized by being environmentally safe, long lasting and effectiveness (AboElyousr and Bagy 2018). Biological control, using beneficial microorganisms, could therefore, be a choice for managing the disease © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. * Correspondence: kaaboelyousr@agr.au.edu.eg Plant Pathology Department, Faculty of Agriculture, Assiut University, Assiut, Egypt Faculty of Meteorology, Env., and Arid Land Agriculture, Department of Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia Full list of author information is available at the end of the article Egyptian Journal of Biological Pest Control Mohamed et al. Egyptian Journal of Biological Pest Control (2020) 30:61 https://doi.org/10.1186/s41938-020-00262-9

resistance Background Bacterial wilt disease caused by Ralstonia solanacearum (Smith) is one of the serious plant diseases worldwide (Peeters et al. 2013). In Egypt, as well as in many countries, it has become a severe problem for the production of potato plant (Abo-Elyousr and Bagy 2018).
Traditional control methods such as short rotation, resistant cultivars, and soil fumigation have been suggested. However, such methods are not always enough effective since R. solanacearum can remain with the infested plant debris in the soil for a long time. Recently, the research focuses on some alternative methods to control the disease that are characterized by being environmentally safe, long lasting and effectiveness (Abo-Elyousr and Bagy 2018).
Biological control, using beneficial microorganisms, could therefore, be a choice for managing the disease (Abd-El-Khair 2020). Many beneficial microbes such as P. fluorescens spp., Acinetobacter spp., Bacillus spp., Bacteriophages, Enterobacter spp., Pseudomonas putida, Paenibacillus macerans, Streptomyces, and Trichoderma spp. have been documented as effective biocontrol agents against R. solanacearum (Ling et al. 2010). Trichoderma spp. have been focused on their antagonistic and mycoparasitic ability to reduce the disease incidence caused by phytopathogens (El-Sharkawya et al. 2018). They play an important role in controlling of soil-borne pathogens, stimulating the plant growth, and enhancing the crop productivity (Sallam et al. 2019). Some of Trichoderma spp. are manufactured and marketed worldwide as commercial biological products, used as soil fertilizers, plant biostimulant, and biofungicide (Woo et al. 2014). Many reports indicated that Trichoderma could induce resistance in plants not only by producing proteins, but also by producing secondary metabolites in the soil amended with decomposed organic fertilizer, which has decreased the R. solanacearum population (Konappa et al. 2018).
The endophytic bacteria are those that could internally colonize plants without induction of any apparent negative effects on the plant morphology and physiology (Gaiero et al. 2013). They are increasingly characterized by their possible use in the phytoremediation, promotion of plant growth, reduction of abiotic stress, and antagonism against many pathogens (Mercado-Blanco and Lugtenberg 2014). Interestingly, wilt disease of some crops such as tomato, caused by R. solanacearum, has been controlled by endophytic bacteria: Pantoea ananatis, Pseudomonas oleovorans, and Enterobacter cloacae (Upreti and Thomas 2015). It was reported that common genera such as Sphingobacterium, Comamonas, Arthrobacter, Curtobacterium, Paenibacillus, Xanthomonas, Serratia, Pantoea, Variovorax Stenotrophomonas, and Enterobacter were frequently isolated from the rhizospheric soil of potato, and many of them were used as potential biocontrol agents (Chamedjeu et al. 2019). For example, Götz et al. (2006) used Enterobacter cowanii as biocontrol agents against bacterial wilt of tomato. E. cloacae was used successfully for controlling many plant diseases such as Fusarium wilt of spinach (Tsuda et al. 2001), bacterial blight of rice (Yang et al. 2000), dry rot of potato (Al-Mughrabi 2010), and damping-off of tomato (Yuliar et al. 2019).
To the best of the authors' knowledge, this is the first time to use E. cloacae and T. asperellum, as a field trial, against R. solanacearum, the causal pathogen of potato wilt. The aim of this study was to investigate the biocontrol efficiency of E. cloacae PS14 and Trichoderma asperellum T34 to manage the wilt disease of potato under field conditions as well as exploring the biochemical responses in relation to defense enzymes that induce the resistance of plants against pathogen.

Bacterial pathogen
R. solanacearum isolate PHYRS3, the causal agent of potato wilt disease, was isolated in a previous work by Bereika (2008). In brief, samples of diseased potato plants showing wilt symptoms were washed 2-3 times by tap water, followed by sterile water and sterilized with 2% sodium hypochlorite solution; the disinfected plant samples were homogenized with 5 ml of sterile 0.05-M potassium phosphate buffer in a sterilized mortar and pestle. A loop of the resulted suspension was streaked onto 2,3,5-triphenyl tetrazolium chloride (TZC) agar medium in 9.0-cm Petri plates and then incubated at 27°C for 48 h and examined daily for bacterial growth. The single colony technique was used to obtain pure cultures of the isolated bacteria by growing on the same media used and kept at 4°C for further studies.

Trichoderma asperellum strain T34
T. asperellum strain T34 was obtained from Biocontrol Technologies S.L. Company Spain and reactivated and maintained on potato dextrose agar (PDA).

Endophytic bacteria
The endophytic bacteria were isolated from healthy potato plants, collected from Assiut Governorate, Egypt, during winter 2016. Segments of potato stems (2 cm) were surface-sterilized, using 2% sodium hypochlorite for 3 min, then with 70% ethanol for 30 s. The segments were rinsed by sterile distilled water 3 times and homogenized in 10 ml acetate buffer (pH 5.2). The homogenized plant tissue was taken by a loop and streaked on the surface of Petri dishes (9 cm) containing nutrient agar medium (NA). The plates were incubated at 28°C for 48 h. Seven bacterial strains were recovered and their pure cultures were kept on NA slants and preserved at 4°C for further use.
Evaluation of antagonistic potentially of the endophytic bacteria against R. solanacearum The antagonistic and pathogenic bacteria were grown individually in 250-ml Erlenmeyer conical flasks containing 100 ml of nutrient sucrose broth and incubated at 28°C for 48 h, and 150 rpm. After incubation, the bacterial growth was centrifuged at 10000×g in sterile microfuge tubes. The supernatants were excluded, and the bacterial cells were collected. Using spectrophotometer (at 600-nm), the bacterial cell density was adjusted to 10 8 cell/ml. The antagonistic activities of seven isolates of the endophytic bacteria were evaluated using the dual culture against R. solanacearum PHYRS3 based on the method described by Abo-Elyousr et al. (2012) with modification. Briefly, a suspension of R. solanacearum PHYRS3 (100 μl at 10 8 cell/ml) was spread over agar surface. After drying, 100 μl of each antagonistic isolate (10 8 cell/ml) were individually pipetted into 5-mm punches in the same agar inoculated with the pathogen. The antibiotic streptomycin (1.0 mg/ml) was used as positive control. After 2 days from incubation at 28°C, the antibacterial effect of the strains was monitored by measuring the diameter of the inhibition zone (mm). The experiment was repeated twice with four replicates for each treatment.

Identification of the potent antagonistic bacterial strain
Based on the above test, the potent antagonistic bacterial strain was selected for identification via 16 s rRNA sequencing according the following protocol.

DNA isolation
The genomic DNA was extracted from the endophytic bacterial strain using the genomic DNA Prep kit (Sol-Gent, Daejeon, Korea) following to the manufacturer's instructions. The extracted DNA was used as a template for PCR to amplify the 16S rRNA gene. Universal bacterial primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used to amplify the nearly complete 16S rRNA gene (Abd-Alla et al. 2012).

PCR amplification
The PCR amplification was performed in a 25-μl reaction volume containing 0.4 μM of each primer, 0.75 U of EF-Taq DNA polymerase (SolGent, Daejeon, Korea), 0.2 mM of each d NTP, 10-50 ng of the template DNA, and 1 × EF-Taq reaction buffer. The thermo cycling conditions included an initial denaturation step at 95°C for 15 min, followed by 30 cycles at 95°C for 20 s, 50°C for 40 s, and 72°C for 1.5 min with a final extension step at 72°C for 5 min. The PCR product was separated by 1.5% agarose gel electrophoresis containing ethidium bromide with buffer of 0.5× Tris-acetate-EDTA (TAE) and visualized using a UV illuminator (Abd-Alla et al. 2012).

DNA sequencing
The PCR product was purified according to the manufacturer's instructions using a SolGent PCR purification kit (SolGent, Daejeon, Korea) (Sanger et al. 1977). The partial 16S rRNA gene sequence was compared to full sequences available in the GenBank database, using a BLAST search (NCBI) for identification of the bacterial strain. Sequences obtained with those retrieved from GenBank database and the sequenced data were deposited in the GenBank under a specific accession number.
Effect of E. cloacae PS14 and T. asperellum T34 on diseases severity under greenhouse conditions The greenhouse trials were conducted at the Experimental Greenhouse of Department Plant Pathology, Assiut, Egypt, during the 2017 growing season. Healthy potato tubers (Solanum tuberosum L.) cv. Berema were surfacesterilized by soaking in 2% sodium hypochlorite for 3 min, washed thoroughly with sterilized distilled water and planted directly in sterilized 25-cm diameter pots. Pots and soil were sterilized by 5% formalin and left for 15 days before planting. Pots filled with 5-kg sterilized sandy-clay soil (3:1 w/w) were kept in the greenhouse under natural temperatures and photoperiods during the growing season. Plants were fertilized every 15 days with (20 g/pots) of 46% urea and irrigated with water when necessary. Forty-five days after planting, potato cultivar was inoculated with R. solanacearum PHYRS3 (the pathogen) suspended in water at 10 8 cell/ml, by using an alcohol knife which was inserted 4-5 cm into the soil to cut the roots along two sides and 20 ml of R. solanacearum PHYRS3 suspension was added to each plant around the basis of each plant (Bereika 2008). Infected control plants were inoculated with the pathogen only and treated with 20 ml sterile distilled water, while healthy control did not receive any treatment (without infection and without treatment). After inoculation, the potato cultivar was kept in a moist chamber at 25°C for 2 days before being transferred to the greenhouse at 25-30°C. Two days after inoculation, 20 ml from each agent suspension (E. cloacae PS14 or T. asperellum T34) was added surrounding the bases of plants. Four replicates were used for each treatment. After 6 weeks, the development of bacterial wilt symptoms was observed. The disease severity was monitored and the percentage of disease severity (DS%) was estimated using the formula suggested by Kempe and Sequeira (1983).

Disease assessment
Disease severity was recorded, using the scale of Kempe and Sequeira (1983) as follows: 0 = no symptoms, 1 = 1-25% of leaves wilted, 2 = 26-50% of leaves wilted, 3 = 51-75% of leaves wilted, 4 = more than 75% and less than 100% of leaves wilted, and 5 = all leaves wilted and died. Disease severity percentage was calculated by the following equation: where "d" is the disease rating on each plant, "d max" is the maximum disease rating possible, and "n" is the total number of plants examined in each replicate.

Application of E. cloacae PS14 and T. asperellum T34 under field conditions
The experiments were carried out at the farm of Faculty of Agriculture, Assiut University, Egypt. Four replicates were used for each treatment and the treatments were distributed in a completely randomized block design. The experimental plot area was 25-m 2 containing 5 rows, each row was 4.5-m in length and the distance between each row was 0.5 m. Potato seed tubers (cv. Berema) were sown on the middle of the ridge at 0.4-m apart. After one month from planting, each treatment was added singly to the plant's soil by drenching around the plants at 20-ml from each microorganism before 48 h of inoculation with R. solanacearum PHYRS3 (10 8 cell/ml) as described in greenhouse experiment. The control plants were treated by 20-ml distilled water after cutting (Winstead and Kelman 1952) and the disease severity was recorded after 6 weeks after inoculation according to Kurabachew and Wydra (2013). At harvest time (110 days after planting), potato plants of 6 plants from each replicate were pulled for measuring a total tuber yield (kg) per replicate and the yield was expressed as ton/hectare.

Biochemical analyses
The effects of E. cloacae PS14 and T. asperellum T34 on biochemical changes of the inoculated potato plants by R. solanacearum PHYRS3 were investigated. Leaves samples were taken at zero time and at 2, 4, 6, and 8 days after inoculation for determination of total phenoland salicylic acid contents and enzyme activities.

Determination of total phenol and salicylic acid contents
One gram of potato plant's leaves was crushed in liquid nitrogen and homogenized in 10-ml of 80% methanol. The homogenate was centrifuged at 10,000 g for 30 min at 4°C. The pellets were wasted after adding ascorbic acid (0.1 g/5 ml). The homogeneous product was evaporated at 65°C in a rotary evaporator and the process was repeated 3 times each for 5 min. The residues were dissolved in 5 ml of 80% methanol. For each treatment, 4 replicates were used (Rapp and Ziegler 1973). The method of Abo-Elyousr et al. (2008) was used to determine phenol content as milligrams gallic acid/gram plant material. Salicylic acid content was estimated, using a modified method by Dat et al. (1998) as micrograms salicylic acid/gram plant material.

Enzymes activity
For determination of activities of peroxidase (PO), polyphenol oxidase (PPO), and lipoxygenase (LO), 1 g of fresh potato plants' leaves was treated with liquid nitrogen and homogenized with 10-ml of 0.1 M Na-acetate buffer (pH 5.2). The mixture was centrifuged at 10,000 g for 30 min at 4°C and the enzyme activity was determined in the supernatants. Four replicates were used for each treatment. Total protein was estimated based on the method described by Bradford (1976) using Bradford reagent spectrophotometrically at 595-nm using Bovine serum albumin as standard.
Peroxidase (PO) activity The enzyme activity was determined spectrophotometrically by the method of Putter (1974), using guaiacol as a substrate. The reaction mixture was composed of 0.2 ml supernatant, 1 ml of 0.1 M Na-acetate-buffer (pH 5.2), 0.2 ml of 1% H 2 O 2 and 0.2 ml of 1% guaiacol. The mixture was incubated for 5 min at 25°C and then measured at 436 nm. The blank was used with extraction buffer. The PO activity was calculated according to the change in absorbance and expressed as enzyme per 1 mg protein.
Polyphenol oxidase (PPO) activity The enzymatic activity was determined, using the method described by Batra and Kuhn (1975). The reaction mixture was 0.5 ml of the supernatant, 2 ml of 50 mM Sorensen phosphate buffer (g/l: KH 2 PO 4 , 6.8; Na 2 HPO 4 ·2H 2 O, 8.99; EDTA, 0.372 and distilled water up to 1000 ml, pH was adjusted to 6.5) and 0.5 ml of the substrate Bren catechol (Sigma Aldrich). The mixture was incubated for 2 h in water bath at 37°C and measured at 410 nm. Activity of PPO = OD at 410 nm/mg protein.

Lipoxygenase (LOX) activity
The enzymatic activity was evaluated according to the protocol of Axelred et al. (1981), the increasing in lipoxygenase activity was measured according to the increase in absorbance at 234-nm resulting from the conjugated double-bond system in the hydroperoxide produced from the substrate, linoleic acid (10-mM sodium linoleate; pH 9). The mixture contained 10 μl of mixture, 20 μl of the substrate, and 1 ml of 50 mM sodium phosphate buffer (pH 6). Absorbance readings were made spectrophotometrically for 3 min at room temperature. A mixture containing buffer and substrate was used as a blank. The activity was measured from the extinction coefficient of 25 mM −1 cm −1 .
Possible mode of action exerted by E. cloacae PS14 and T. asperellum T34 Salicylic acid production Salicylic acid (SA) produced by E. cloacae PS14 and T. asperellum T34 were determined, following the method described by Meyer and Abdallah (1978). The microorganisms were grown in the standard succinate medium at 28°C for 48-h components from K 2 HPO 4 (11.5 g/l), (NH 4 ) 2 SO 4 (28.7 g/l), and distilled water up to 1000 ml. The pH was adjusted to 7.0 with 20% NaOH prior to autoclaving. Cells were collected by centrifugation at 8000×g for 5 min and re-suspended in 1 ml of 0.1 M phosphate buffer. A 4-ml of cell-free culture supernatant was acidified with 1-N HCl to pH 2.0 and SA was extracted in CHCl 3 . Four milliliters of water and 5 μl of 2-M FeCl 2 were added to the pooled CHCl 3 phases. The absorbance of the purple iron-SA complex, developed in the aqueous phase, was read at 527 nm. A standard curve was prepared with SA dissolved in succinate medium and quantity of SA produced was calculated.
Indole acetic acid production E. cloacae PS14 and T. asperellum T34 were inoculated by TSB with tryptophan as a precursor (100 μg/ml) on a shaker for 30 min. The culture's supernatants were collected after centrifugation for 10 min at 2000 rpm and 1 ml of cell-free culture filtrate was mixed with 2 ml of Salkowski reagent (1 ml of 0.5 M FeCl in 50 ml of 35% perchloric acid) and incubated at 28°C for 30 min. Quantification was done calorimetrically at 530 nm comparing to indole acetic acid production (IAA) standard curve (Nandhini et al. 2012).

Quantitative detection of hydrogen cyanide and siderophore
The antagonistic microorganisms were grown in trypticase soy broth (TSB, Hi media, India). Filter paper was cut into uniform strips of 10-cm-long and 0.5-cm-wide saturated with alkaline picrate solution and placed inside the test tubes in hanging positions. After incubation for 48 h at 28°C, the sodium picrate in the filter paper was reduced to a reddish compound in proportion to the quantity of hydrocyanic acid evolved. The color was eluted by placing the filter paper in a clean glass test tube containing 10 ml of distilled water and absorbance was measured at 625 nm (Sadasivam and Manickam 1992).
The quantitative estimation of siderophore produced by bioagents was done by CAS-shuttle assay (Schwyn and Neilands 1987). The strains were grown in a succinate medium and incubated at 28°C for 24 to 30 h and shaking at 120 rpm. After incubation, the fermented broth was taken and centrifuged at 10,000×g for 10 min at 4°C and the cell-free supernatant was mixed by 0.5 ml CAS solution. The color obtained was determined at 630 nm after 20 min from incubation with reference containing 0.5 ml CAS solution with 0.5 ml uninoculated succinate medium. The percentage of siderophore units was determined as the proportion of CAS color shifted using the formula% Siderophore units = (A r − A s /A r ) × 100, where A r = Absorbance of reference at 630 nm (CAS reagent) and A S = Absorbance of sample at 63 nm

Statistical analysis
Data were initially examined for their normal distribution of errors using Shapiro-Wilk's W test and for homogeneity of variances using Levene's test. All statistical analyses were performed with SPSS 22.0 software.
The data were analyzed for significance of variation using one-way analysis of variance (ANOVA) for antagonistic capability and for the rest of experiments twoway analysis were used. The least significant difference (LSD) test was used at P < 0.05 to identify the significant differences among the means of the treatments according to Gomez and Gomez (1984).

Results and discussion
Antagonistic activity and identification of the endophyte E. cloacae PS14 The antagonistic activity of 7 bacterial isolates against R. solanacearum PHYRS3 was evaluated, using dual culture method ( Table 1). The results indicated that the highest inhibitory effect against the PHYRS3 was attributed to the bacterial strain no. 7, which caused production of inhibition zone as 16.9 mm compared with 20.0 mm because of specific antibiotic streptomycin. The potent antagonistic bacterium was identified by 16S rRNA gene and their sequences were compared to data available in the GenBank using BLAST search. The results indicated that the bacterial strain belongs to the genus Enterobacter and it could be identified as E. cloacae (Table 2) with a similar, percentage as 86.22%. It was given a specific strain number as E. cloacae PS14 and it was allocated in the GenBank under accession number Mn385618. The findings could be supported by those of Upreti and Thomas (2015) who isolated E. cloacae, Pantoea ananatis, and Pseudomonas oleovorans as endophytic bacteria from different tomato cultivars. However, a relatively low similarity percentage of the strain identity with those allocated in the GenBank could support our hypothesis that it is a new strain of E. cloacae that has specific characteristics such as its antagonistic potency and biological control efficiency against the target pathogen. The use of plant-associated microorganisms in biological control of plant diseases was mentioned as an efficient and ecofriendly method (Xue et al. 2009). The suppressive effect of the antagonistic bacterium against the pathogen could be mainly due to its antibiosis activity, where it has the ability to synthesize certain molecules to inhibit the pathogen development that was indicated by an inhibition zone around their growth (Chamedjeu et al. 2019).
Efficiency of E. cloacae PS14 and T. asperellum T34 in controlling the wilt of potato under greenhouse and field conditions The results illustrated in Figs. 1 and 2 showed that the antagonists, E. cloacae PS14, and T. asperellum T34 significantly decreased the disease severity of wilt disease than the control (infected plants with the pathogen only) under greenhouse conditions. The disease severity was reduced by 10.73 and 26.50% due to the 2 respective bioagents, compared to 93.25% due to the pathogenic bacterium. Under field conditions, the bioagents successfully protected the potato plants against the destructive effect of the pathogen (Table 3). The disease severity was reduced by 39.00-49.06% from the infected control as the result of application of microorganisms, during the 2 seasons of the experiment. The used of both agents caused increasing the productivity of the crop by 20.44-40.96% relative to the control during the 2 seasons. Obtained results are somewhat in agreement with those of Upreti and Thomas (2015), who used P. oleovorans, Pantoea ananatis, and E. cloacae to manage the disease caused by R. solanacearum on tomato plants. These authors mentioned that the presence of endophytic bacteria, that have antagonistic potentiality against pathogenic organisms, could reveal a possible role of endophytes associated with the root in natural defense against the pathogens. Also, Podolich et al. (2015) reported that endophytic bacteria may play an important role in crop protection by stimulating their active form in response to environmental stress or pathogen attack as well as improving the growth and plant health.
Effect of E. cloacae PS14 and T. asperellum T34 on production of biochemical precursors causing resistance's induction in potato plant

Phenol compounds
Salicylic acid Results showed a significant increase in salicylic acid content in the inoculated potato plants with T. asperellum T34 and E. cloacae PS14 than either only infected control or healthy plants (Fig. 3). The SA accumulation increased, 2 days after inoculation of plants, with the bioagents until the 8th day and SA content in plants, treated with T. asperellum, was higher than those treated with E. cloacae. The increase in SA content in the treated plants confirms the hypothesis that both rhizobacteria (PGPR) and Trichoderma spp. could stimulate the plant resistance, in addition to their suppressive effect on the phytopathogens. It was confirmed that these microorganisms were able to stimulate plant growth by mediating the plant hormones ethylene (ET) and induction of the resistance in plants through production of salicylic acid (SA) and jasmonic acid (JA) (Hermosa et al. 2012). Hassan and Abo-Elyousr (2013) suggested that SA accumulation was essential for expression of multiple modes of plant disease resistance. Because SA is a plant defense phenolic compound against pathogens, it should be detected in both infected and distal leaves in response to pathogen attacks. Obtained results confirm that enhanced SA contents are, thus, a prerequisite for expression systemic acquired resistance against R. solanacearum in potato plants.
Total phenol content The total phenol contents in the inoculated potato plants with the pathogen and treated with T. asperellum T34 and E. cloacae PS14 were significantly higher than that in only infected plants (control) or healthy plants (Fig. 4). The total phenol content in plants, treated with the 2 agents began to increase after the first 2 days from inoculation until the 6th day and then the concentrations began to decrease, however, the increase was not significantly different. These results agree with those of Vinayarani and Prakash (2018) who reported that the accumulation of phenolics was observed in turmeric rhizomes treated with T. asperellum after the 2nd from inoculation with the Pythium aphanidermatum and the maximum accumulation was observed on the 5th day after inoculation (Vinayarani and Prakash 2018). Khan and Bano (2019) mentioned that treatment of wheat plants with PGPR bacteria increased  Values in the same column followed by the same letter(s) are not significantly different as determined by the LSD test (P ≤ 0.05) the phenolic content. Accumulation of phenolic compounds in response to an infection in plants was reported by Hammerbacher et al. (2011) who confirmed that phenolic compounds are produced through the phenylpropanoid pathway and/or rapid translocation and modification of existing compounds. The accumulation of phenolic compounds, in chili plants, at the site of the infection with anthracnose disease was correlated with the limitation of pathogen development, since these compounds are toxic to pathogens (Jayapala et al. 2019). Resistance may also be increased by changing the pH of plant cell cytoplasm due to an increase in phenolic acid content that inhibits of development of pathogen (Bereika 2008;Hassan and Abo-Elyousr 2013).

Enzymatic activities
Peroxidase (PO) activity The results displayed that T. asperellum T34 significantly induced the maximum levels of PO activity in potato leaves infected with pathogen on the 8th day after inoculation than the either infected or heathy plants (Fig. 5). However, E. cloacae PS14 showed the highest level of PO activity in potato leaves on the 4th day after inoculation. This finding is supported by several reports that mentioned the enhanced PO activity in plants, when they were subjected to fungal, bacterial, and viral infections (Safdarpour and Khodakaramain 2018). Peroxidase caused lignin biosynthesis that provides a physical barrier and/or limits the degree of pathogen invasion and spread throughout the plant (Vidhyasekaran 2008). POX increases due to systemic resistance, a rapid synthesis of reactive oxygen derivatives by oxidative burst contributes to cell death and prevents pathogenic activity (Prasannath et al. 2014).
Polyphenol oxidase (PPO) activity Treatment of potato plants with T. asperellum T34 and E. cloacae PS14, after infection with the pathogen, induced the PPO activity in the potato plants in varied levels (Fig. 6). After 2 days from inoculation, potato plants treated with T. asperellum T34 exhibited a significant gradual increase in PPO activity until the 8th day than the only infected or healthy plants. E. cloacae PS14 showed a high level of PPO activity from the 2nd until the 4th day, and then PPO decreased on the 6th and 8th days than the only infected plants. The significance of PPO activity in disease resistance probably be due to its property of oxidizing phenolic compounds to quinines, it is often more toxic to microorganisms than the original phenol (Safdarpour and Khodakaramain 2018). Generally, toxicity of PPO-created quinones to pathogens and plant cells, inducing cell death, alkylation and decreased bioavailability of cell proteins to the pathogen, crosslinking protein or other phenolic quinones, forming a physical barrier to cell wall pathogens and quinone redox cycling leading to H 2 O 2 and other reactive oxygen specificities (Konappa et al. 2018).
Lipoxygenase (LOX) activity T. asperellum T34 and E. cloacae PS14 significantly increased the activity of lipoxygenase (LOX) in potato leaves infected with the pathogen than either only infected or healthy plants (Fig. 7). Increase in LOX level began after 2 days from inoculation of plant with the bioagents till 8 days; however, T. asperellum T34 showed higher activity of LOX than E. cloacae PS14. The results agree with those of Kurabachew and Wydra (2013). This might be due to the fact that pathogen inoculation does not affect linoleate-consuming LOX. In potato plants, which could be due to an increase in the level of transcription of the two isoforms of LOX: Tomlox D and Tomlox E. LOX products contribute to the defense reaction in plants-pathogen interaction by inhibiting pathogen growth, phytoalexin accumulation, and signal transduction (Mariutto et al. 2011). In vitro production of siderophore, HCN, IAA, and SA by E. cloacae PS14 and T. asperellum T34 to confirm their possible mode of action Both microorganism, E. cloacae PS14 and T. asperellum T34, were able to produce a considerable amount of siderophore, HCN, IAA, and SA (Table 4). The endophytic bacterial E. cloacae PS14 produced a higher amount of siderophore, HCN, and SA than T. asperellum T34; however, the situation was reversed in the case of IAA. E. cloacae PS14 produced 0.884, 3.61, and 1.488 μg/ml of siderophore, HCN, and SA, respectively. T. asperellum T34 produced 0.225 μg/ml of IAA. Our findings could be supported by those of Calin et al. (2019) who reported that T. asperellum had the ability to produce IAA. That is recognized as the key plant growthpromoting substance regulating plant physiological and developmental functions (Srisuk et al. 2018). Trichoderma spp. can produce IAA with varied amounts (Mohiddin et al. 2017). In other work, E. cloacae strain OS03 had the ability to produce IAA in varying levels in the medium containing L-tryptophan than under a cop of physicochemical conditions (Panigrahia et al. 2019). Sheng et al. (2008) mentioned that E. cloacae could have a significant impact on the growth of plants, in particular by producing IAA (Panigrahia et al. 2019). Siderophores are iron chelating compounds that have a serious part in the field such as agriculture, bioremediation, biosensor, and medicine (Venkat et al. 2017). In a similar report to ours, E. cloacae strain PGLO9 exhibited activity of siderophore production and showed a positive antagonistic ability to phytopathogens (Verma et al. 2018).
Trichoderma can produce siderophores to control plant disease and promote growth opportunities to replace chemical pesticides (Calin et al. 2019). Siddiqui et al. (2006) reported that HCN production is an important feature found in different microorganisms, since it indirectly promotes plant growth by controlling certain soilborne diseases (Siddiqui et al. 2006). Positive correlation has been documented between production of HCN in vitro and plant protection and their defense system. It was thought that HCN direct inhibition of fungi was one of the major mechanisms of action. There are numerous reports on biocontrol activity of HCN production in the rhizosphere in tobacco plants. Also, it can stimulate the root length and root hair germination (Ghosh et al. 2018). Also, specific strain of E. cloacae (K2 strain) was reported as a good HCN-producer (Pramanik et al. 2018). T. asperellum-cucumber interaction increased SA and peroxidase activity as an induction response in the plants. In addition, after inoculation of Trichoderma into the plants, a systemic resistance of plants increased as the increase in SA and JA rates were observed (Hermosa et al. 2012). Also, endophytic bacteria increased the growth of sunflower seedlings and inhibited the growth of pathogenic fungi by developing SA (Klessig et al. 2016).

Conclusion
The results conclude that the new endophytic strain of E. cloacae PS14 was a potent biocontrol agent against R. solanacearum-the causal pathogen of wilt of potato. The use of T. asperellum T34 and E. cloacae PS14 successfully suppressed the R. solanacearum in vitro, in vivo, and in situ. It caused reduction of the disease severity and increased the crop yield in a level close to T. asperellum T34. The main mechanism of action of the new bacterial strain was due to production of antibacterial agent as well as induction of systemic resistance of potato plant. Successful application of the new bacterial strain on the field scale for two successive seasons encourages its application on a large scale in biological control of plant disease, and further research to find an appropriate formulation and approving application of the new strain for a wide range of plant diseases was recommended.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Evaluation of simultaneous incidence of head space and temperature on biochemical methane potential in food waste

Abstract The anaerobic digestion (AD) of food waste (FW) has been recognised as a low-cost and high-efficiency alternative that mitigates the impacts of the final disposal of FW, while facilitating the production of high value-added by-products, such as biogas and digestate. In this study, the simultaneous incidence of two significant variables in the AD of FW were evaluated: the temperature (20, 35 and 55 °C), which affects the kinetics of the process, and the head space (20, 35 and 50%), which can affect the pH and therefore the biochemical methane potential (BMP). A composite central design was used, and response surface methodology (RSM) and kinetic models (the first-order and Gompertz models) were used to analyse the process. At higher temperatures and smaller head spaces, inhibition phenomena were reduced by the consumption of bicarbonate alkalinity and decreasing the pH, resulting in a higher process performance. The optimal conditions were a temperature of 48.9°C and a head space of 20%. The Gompertz kinetic model fit the process dynamics better and with a lower error than the first-order model.

ABOUT THE AUTHORS Miguel R. Casallas-Ojeda He is an Environmental Engineer, a student of master in engineering, emphasis Sanitary and environmental engineering of the Universidad of Valle. He is also a member of the research group Study and Control of Environmental Pollution (ECCA).
Luis Fernando Marmolejo-Rebellón He is a Sanitary Engineer and a PhD in Engineering from Universidad of Valle. He is an associate professor at the School of Natural Resources and Environment Engineering at Universidad of Valle. Also, member of the Pollution Study and Control research group Environmental (ECCA).
Patricia Torres-Lozada She is Ing. Sanitaria, MSc. and PhD. in Civil Engineer for University of Sao Paulo. She is a full professor at the School of Natural Resources and Environment Engineering at Universidad of Valle and director of the Research and Control Group of Environmental Pollution.

PUBLIC INTEREST STATEMENT
A consequence of growing population, is the production of Municipal Solid Waste and due to their composition predominantly organic (as Biowaste and Food Waste), the anaerobic digestion is a biological alternative that not only allows to increase the useful life of landfills and minimize the generation of leachate and GHG emissions, but also its valorization for the production of energies such as methane. Biochemical Potential of Methane (BMP) assay, is the main tool of prediction, on controlled scale of the potential energetic of the substrate. Both, the head space and the temperature have influence the process and the methane generation, and is necessary to study their join influence, to stablish the optimal condition for the process.
Final disposal (FD) in landfills (LF) is not an appropriate management strategy for MSW (Saer, Lansing, Davitt, & Graves, 2013;Schott, Wenzel, & la Cour Jansen, 2016) because the subsequent environmental impacts, such as the generation of greenhouse gases (GHG) and leachates, however, LF remains the most commonly used management alternative for MBW and FW in most developing countries, such as Colombia, which is covered by Decree 2820 (2010) from the Ministry of Environment, Housing and Territorial Development (MAVDT 2010). Silpa, Yao, Bhada-Tata, and Van Woerden (2018) have reported that the GHG produced worldwide from MSW disposal in LF are on the order of 1.6 × 10 9 tons of carbon dioxide (CO 2equivalent ), representing 5% of global emissions, which deteriorate the ozone layer and negatively impact human health and the environment (Christensen, Manfredi, & Knox, 2011). This form of management also has other unfavourable effects, such as the depletion of the useful life of the FD sites, which are in increasingly limited supply (Manyoma, Orejuela, Torres, Marmolejo, & Vidal, 2015).
However, being predominantly organic, these residues have a high use potential through strategies such as anaerobic digestion (AD) and composting. AD is a biochemical process that occurs in the absence of molecular oxygen and consists of multiple stages involving different microorganisms; the process occurs via the transformation of complex compounds, such as carbohydrates, proteins and lipids, into simpler products such as 90% biogas (mainly methane -CH 4 , which has a high energy potential) and 10% biosolids or digestate (Foresti, Florêncio, Van Haandel, Zaiat, & Cavalcanti, 1999;Satchwell et al., 2018), which has a high potential for use in soil recovery and agricultural activities (Tampio, Salo, & Rintala, 2016). Thus, research on AD technology has increased in recent years (Lin, Xu, Ge, & Li, 2018).
In this study, the simultaneous incidence of temperature and head space on the BMP of FW was evaluated. The FW simulated the characteristics of MBW from a locality that performs separation at the source and selective MSW collection, which are fundamental premises of the use of this type of substrate (Han & Zhang, 2017;Lakeh et al., 2019;Razavi, Koupaie, Azizi, Hafez, & Elbeshbishy, 2019;Soto-Paz et al., 2019).

Substrate and inoculum
A sample was reconstituted with the characteristics of the MBW from a locality that performs separation at the source and selective collection of MSW. The substrate was formed from raw FW that was separated at the source of a university restaurant that serves an average 4338 lunches per day  and a market place in the City of Cali, Colombia. The FW composition was as follows: carbohydrates (55.4%), citrus fruits (25.0%), fibres and minerals (8.2%), non-citrus fruits (8.2%), herbs and others (3.2%) Soto-Paz et al., 2019).
An inoculum was formed using a 75/25 (V/V) mixture of flocculent sludge and granular sludge, respectively. The first one was obtained from an anaerobic digester of a municipal wastewater treatment plant (WWTP) that serves a population of more than two million inhabitants, and the granular sludge was obtained from an anaerobic reactor of a slaughter plant for cattle and pigs (Parra-Orobio et al., 2018).

Calculation of theoretical methane potential
Different calculation schemes were used to compare the theoretical methane production with the experimental results. In the first calculation, the stoichiometry of the substrate degradation reaction was used with Equations 1 and 2 (Buswell &Muller, 1952 andSosnowski et al., 2003). A second calculation (Equation 3) was performed using the percentage of carbohydrates, proteins and lipids in the sample (Lesteur et al., 2010;Raposo et al., 2012 ;Nielfa, Cano, & Fdz-Polanco, 2015).
where the number of moles of carbon, hydrogen, oxygen and nitrogen are represented by n, a, b and c, respectively; 22.4 is the volume in litres (L) occupied by an ideal gas at standard conditions; 1000 corresponds to the conversion factor from L to mL; and 12, 1, 16 and 14 are the molecular weights of carbon, hydrogen, oxygen and nitrogen, respectively.

Experimental design
A composite central experimental design was performed in which the simultaneous incidence of the temperature (X 1 ) and the head space (X 2 ) were evaluated using response surface methodology (RSM) (Montgomery, Runger, & Hubele, 2009). In this study, each factor was evaluated at three levels corresponding to minimum, intermediate and maximum values (the normalised values were −1, 0, +1, respectively), following studies by Zhang and Jahng (2012) Table 1 presents the values used in the design and the different experimental configurations. The BMP was the response variable.

Experimental setups and process monitoring
A CB15 industrial food processor was used to grind the substrate to a particle size of less than 30 mm (Izumi et al., 2010;Pearse et al., 2018), with which the PBM assays were developed. A substrate/inoculum (S/I) ratio of 1 gVS substrate /gVS inoculum , was maintained, acording Parra-Orobio et al., (2018).
The BMP assays were performed in triplicate in 0.25 L amber glass reactors. To ensure that the manometric measurement of the biogas corresponded mainly to methane, a polypropylene cartridge containing four NaOH beads was placed inside each bottle under the lid to capture CO 2 (Holliger et al., 2016;Souto, Aquino, Silva, & Chernicharo, 2010). A manometer (WIKA, model CPG500) was used to record the pressure of the biogas that accumulated in the headspaces of the reactors. The biogas composition was confirmed by chromatography (Shimadzu GC-2014). Nutrient addition was performed, and a control reactor (target) containing only the inoculum and distilled water was used to subtract endogenous production (Cárdenas-Cleves et al., 2016).
The temperature was controlled by maintaining the lowest temperature (20 ± 1°C) in a WTW TS 606/G12-i incubator; the intermediate temperature (35 ± 0.5°C) in a WTW TS 606-G/2-i incubator and the high temperature (55°C ± 1.5°C) in a WTB binder E-28 oven. The BMP was determined using equations formulated by Aquino (2007), Giménez, Martí, Ferrer, and Seco (2012) and Cárdenas-Cleves et al. (2016), where the proportion of the dissolved methane was used to determine the volume of methane produced under standard conditions. The assays were manually agitated and monitored four times a day , and biogas samples were withdrawn for chromatographic analysis. The monitoring was terminated upon the observation of asymptotic behaviour, following the recommendations of Holliger et al. (2016).

Data processing
The results of the characterisation of the substrate and inoculum were analysed using descriptive statistics tools. The production results obtained from each of the experimental configurations were  processed using MATLAB® 2017 software to develop a response surface, showing the optimal points at which the variable of interest (the BMP) was maximised. A two-way ANOVA model was similarly implemented, whereby a second-degree polynomial (Equation 4) was generated to reflect the interaction of the considered variables in the process (Zainudin et al., al., 2005). The data were normalised using the normal standard variation method, as described by Equation 5: where Y is the response variable; b 0 is a constant coefficient; b i is the linear coefficient; b ij is the coefficient for the interaction of the variables; b ii is the quadratic coefficient; x i and y i are the coded values of the variables; Xi is the intercept of each variable ( Figure 2); x is the average of the selected values; and σ is the deviation from the selected values (the selected values are presented in Table 1).

Process kinetics
The experimental data were fitted to the kinetic models presented in Table 2.
The mean squared error (MSE) was determined for the model fits. The kinetic analysis was performed in the MATLAB® 2017 software using the Levenberg-Marquardt algorithm (Güçlü, Yılmaz, & Ozkan-Yucel, 2011). Table 3 presents the results of the physico-chemical characterisation of the substrate and the inoculum.

Characterisation of substrate and inoculum
The pH of the FW is within the reported range for this type of substrate and is consistent with characteristic BA and VFA concentrations of easily acidifiable substrates, showing the need for an external alkalising source (Steinmetz et al., 2016;. Although the COD, TOC and VS/TS ratio indicate a high content of organic matter, the COD filtered /COD total ratio confirms the predominance of particulate organic matter, which can be a limiting condition in hydrolysis (Li et al., 2012;Aldin et al., 2011). The TN and TP values show that nutrients are provided for the requirements of the microorganisms involved in the biological process and reflect the presence of legumes in the FW (Elser, 2012). The values for lignin and cellulose (which are slowly degrading organic materials) are similar to those from other studies on AD (Parra-Orobio et al., 2018) and composting (Soto-Paz et al., 2019) of this type of waste, which was separated at the source and obtained by selective collection.
The pH and the BA/TA ratio (0.63) of the inoculum indicate a good buffer capacity, which prevents the accumulation of VFAs produced during AD. These values are similar to previously reported values, such as by de Raposo et al., (2012) and Torres-Lozada, Díaz-Granados, and Parra-Orobio (2015). Although the VS/TS ratio (0.53) confirms that the incorporation of granular sludge into flocculent sludge improves the characteristics of the inoculum, the SMA of 0.15 gCOD CH4 /gVS is low, while being within the range reported by Angelidaki et al.

Theoretical potential of methane
The elemental analysis and composition of the FW samples, in terms of carbohydrates, proteins and lipids (Table 3), was used to determine the theoretical production using Equations 1, 2 and 3. Considering the reaction stoichiometry in Equations 1 and 2 results in a theoretical production of 429 mLCH 4 /gVS; however, these are theoretical methods based on an elemental analysis, in which 100% degradation of the FW is assumed (Fernandez et al., 2008), which corresponds to an ideal theoretical condition. For example, with respect to carbon, the degraded fraction corresponds only to anaerobically biodegradable organic carbon, which is also affected by the presence of materials such as lignin and cellulose (Puyuelo, Ponsá, Gea, & Sánchez, 2011). Some components, while being organic in nature, are difficult to degrade and have limited solubility and as it was shown in the physico-chemical characterisationand of the substrate, they represent an important fraction in these residues. Using Equation 3 resulted in a production of 127 mL CH4 /gVS, which is close to the maximum value of 107 mLCH 4 /gVS obtained in the present study.
By contrast, Equation 3 only accounts for easily degradable monomers in the FW (Mahmoud et al., 2018). Nielfa et al. (2015) have reported an error of only 10% for the experimental production using Equation 3. Thus, different AD incidence variables need to be simultaneously evaluated for FW to better correspond the theoretical conditions, while remaining cognisant that not all of the substrate may be degraded or easily assimilated.
10. Response surface methodology (RSM) for determination of optimal temperature and head space Figure 1 shows the response surfaces for the interaction between (a) the head space and time; (b) the temperature and time; and (c) the interaction of the two considered variables. Figure 1(a) shows an inverse relationship between the head space and methane production, which is in agreements with the results of Stumm and Morgan (2012) and Himanshu, Voelklein, Murphy, Grant, and O'Kiely (2017), wherein increasing the reactor head space was found to decrease the methane yield, because the pH and the partial pressure of CO 2 are related to the presence of CO 2 in the biogas and the inorganic carbon dissolved in the interior of the reactor. Rittman and McCarty (2001) and Panigrahi and Dubey (2019) reported that increasing the partial pressure (the lower head space) increases the dissolution of inorganic carbon, which is a natural buffering agent and prevents microbial populations (i.e. methanogenic Archaea) from being affected by decreases in the pH. Microbes play a critical role in the degradation process and the transformation of intermediate products to methane. Some of these microbes are highly pH-sensitive, and some bacterial consortia require an almost neutral pH to perform well (Jankowska et al., 2015). Figure 1(b) shows that thermophilic temperatures result in approximately 50% more methane production than mesophilic temperatures, which is in agreement with reports by Kim et al. (2006) and Mirmohamadsadeghi et al. (2019). High temperatures also favour the solubilisation of this type of compound, facilitating degradation by microorganisms (Frigon & Guiot, 2010;Shi, Guo, Zuo, Wang, & Zhang, 2018), which increases methane production, in contrast to mesophilic or psychrophilic conditions, which lower production.
Figure 1(c) shows the interaction between the temperature and the head space. The area delimited by temperatures between 50 and 55°C and head spaces between 20 and 25% correspond to the conditions under which the BMP increased in this study. The optimal values for each variable were determined by using Equation 5 to denormalise the values and data from the intercepts in Figure 2. This analysis shows that the optimal conditions are a temperature of 48.9°C and a head space of 20%, which is consistent with the aforementioned results and reports by Deepanraj et al. (2015) and Shi et al. (2018) of increased methane production under thermophilic temperature conditions (50-55°C). The ANOVA analysis was fitted to a 2FI quadratic interaction model for the interaction of two variables, which has been widely used for authors as Li et al. (2015) and Oliveira, Alves, and Costa (2015). The analysis produced Equation 8 with a R 2 value of 0.98 and p = 0.02: y ¼ 65:05 þ 25:15 Ã T À 14:16 Ã Hs À 5:42 Ã T Ã Hs (8) where y is the response variable (BMP), T is the temperature, and Hs is the head space.
Equation 8 shows that temperature variable has the most significant impact on the process, with a coefficient of 25.15 compared to the head space coefficient of −14.16. The coefficient of −5.42 for the interaction between the two variables is less significant. Thus, at higher temperatures, the BMP increases, and the greater the head space is, the lower the BMP. This result is consistent with the significance of each variable: p < 0.0001 (temperature), p = 0.0002 (head space) and p = 0.0290 (interaction of the two variables), where the interaction of the temperature and the head space has the p-value closest to 0.05.
Note that although the degradation rate over the thermophilic range is generally faster than over the psychrophilic or mesophilic ranges, there are disadvantages associated with the thermophilic range. i. Instabilities are introduced into the process from temperature fluctuations, along with the risk of accumulation of VFAs (i.e. propionic acid) from an enhanced high reaction rate, especially during acidogenesis (Mao, Feng, Wang, & Ren, 2015;Panigrahi & Dubey, 2019;Rajagopal, Bellavance, & Rahaman, 2017). ii. More energy must be expended to maintain the temperature above ambient (Shi et al., 2018). Thus, the cost/benefit ratio for production versus the required consumption should be evaluated to determine the feasibility of the proposed strategy. Most AD processes for FW, including industrial scale processes, are carried out under mesophilic conditions to ensure that the process remains stable under the high organic loads supplied .
Given the above-mentioned limitations of implementing thermophilic conditions and increasing the BMP without incurring cost overruns for reactor heating, it is advisable to explore alternatives, such as i. the use of insulating materials for temperature maintenance (Martí-Herrero, Soria-Castellón, Diaz-de-Basurto, Alvarez, & Chemisana, 2019); ii. a staged process or phase separation (Wu, Kobayashi, Li, & Xu, 2015;Xiao et al., 2018); iii. pre-treatment of the substrate and/or inoculum (Li et al., 2016;Parra-Orobio et al., 2017) and iv. innovative studies on the interaction between variables to optimise the process to achieve high biogas production at the lowest possible expense. Table 4 shows the kinetic parameters obtained using the first-order and Gompertz models, along with the VFAs obtained for each configuration at the end of the process.

Kinetic analysis of process
Using the Gompertz model resulted in a maximum production of 133.45 mLCH4/gVS (G), which is far from the maximum production (I) calculated using the first-order model (348.36 mLCH4/gVS), showing that the first-order model overestimates production (Díaz et al., 2015). The theoretical value of 127 mLCH4/gVS obtained using the substrate composition (in terms of the carbohydrates,   (Nielfa et al., 2015).
Following Donoso-Bravo, Pérez-Elvira, and Fdz-Polanco (2010) and , R 2 should be maximised and the error (RMSE) should be minimised. The Gompertz model produced the best fit to the data (R 2 = 0.99 and RMSE < 2.12) and thus describes the process better than the first-order model. A comparison of the kinetic parameters shows that the first-order model, despite its widespread use, did not adequately estimate the methane production for all of the experimental configurations (Díaz, Reyes, Lundin, & Horváth, 2011) and that the Gompertz model better represented the data, in agreement with the results of Cárdenas-Cleves, Marmolejo-Rebellón, and Torres-Lozada (2018). The latency phase increased with the head space (Table 4), indicating the impact of this parameter on methane production via the accumulation of VFAs and the absence of CO 2 dilution because of insufficient pressure (Valero et al., 2016).
The VFA concentrations are consistent with the high presence of acidogenic bacteria (AAE , Table 3), which transform organic matter into VFAs such as valeric, propionic and butyric acids . This bacterial presence, in conjunction with the bacterial growth rate (Rmax), explains the high VFA concentrations at the end of the process. In addition, methanogenic Archaea are known to be very sensitive to changes in pH, requiring almost neutral conditions (Jankowska et al., 2015). The VFA concentrations at the end of the process showed that a neutral environment did not predominate in most of the assays, which is in agreement with the experimental methane production.
The results from Table 4 for the latency phase of the process (λ) show that a short latency does not always favour AD, because increasing the reaction rate or the degradation of VS also increases the accumulation of VFAs, which can inhibit the process if there is no buffer capacity (Lee et al., 2015). By contrast, a prolonged latency phase may allow the microbial consortia to better adapt to the substrate and increase methane production. These results were reflected in the D and G assays. These assemblies were subjected to different temperatures for the same head space value, and different yields were obtained for different latency phase values.
The first-order model does not take into account the latency phase and overestimates methane production data. However, this model is useful for describing hydrolysis in the process: the data showed that the reaction speed was generally not rapid, except in the G assay . Inhibition phenomena may have occurred in the assays with head spaces above 20%, but at lower rates than at the mesophilic and thermophilic temperatures, especially given the reaction rates at elevated temperatures (Mirmohamadsadeghi et al., 2019). Thus, the incidence of the head space is reflected in Rmax, which increases with the temperature.
In general, it can be stated that the Gompertz model is a non-linear model that can be used to evaluate the entire process easily, whereas the first-order model, as its name indicates, reflects the effect that the variable of interest has on hydrolysis. These models are not comparable but can be complementary: the Gompertz model can be used to provide data on the latency phase, whereas the first-order model can provide information on the critical stage of AD of FW (Rajagopal et al., 2017).

Conclusions
• FW contains lignocellulosic compounds that degrade slowly and can thus inhibit high AD yields. Equations have been developed to estimate the potential of methane production from this type of substrate.
• An ANOVA of the considered variables shows that the temperature is the highest incidence variable in the process. The optimal temperature was found to be 48.9°C, and temperatures above this value did not result in increased methane production. The optimal head space was found to be 20%, and head spaces greater than this value resulted in the inhibition of the process from the accumulation of VFAs.
• The result of the models implemented in the study showed that the Gompertz model best describes the AD of FW because the latency phase of the process is considered and does not overestimate methane production, unlike the first-order model. The two models are not comparable to each other.


extfieldsofstudy: Medicine Biology

text:
Attenuation of a recombinant Marek's disease virus lacking the meq oncogene and evaluation on its immune efficacy against Marek's disease virus

SC9-2 is a recombinant Marek's disease virus (MDV) strain lacking the meq oncogene. Previous study demonstrated that SC9-2 virus provides good protection against challenge with a very virulent MDV rMd5, but it induces immunosuppressive effects in specific pathogen-free (SPF) chickens. In the present study, SC9-2 was serially passaged on chicken embryo fibroblast (CEF) cell cultures. The pathogenicity and immune efficacy of SC9-2/10th and SC9-2/40th against rMd5 were evaluated. Animal experimental results showed that SC9-2/10th and SC9-2/40th showed no lethality or tumorigenicity in SPF chickens. Body weight of chickens inoculated with SC9-2/40th were significantly higher than that of the chickens inoculated with SC9-2/10th but lower than that of the uninoculated controls. The severity of bursa and thymus atrophy (BTA) and spleen enlargement in SC9-2/40th-inoculated chickens were also weaker than the SC9-2/10th-inoculated ones but stronger than the uninoculated controls. Chickens inoculated with SC9-2/40th and SC9-2/10th showed similar antibody levels induced by H9N2 subtype avian influenza virus/Newcastle disease virus inactivated vaccines, both of which were lower than the uninoculated controls. Replication of SC9-2/40th was significantly lower than SC9-2/10th in feather follicle epithelium (FFE) of infected chickens. The immune protection index of SC9-2/40th was also lower than that of SC9-2/10th, but the difference was not significantly, and both of which were significant higher than that of the commercial MDV vaccine CVI988/Rispens. The results of our studies demonstrated that SC9-2/40th showed weaker severity of BTA, spleen enlargement, and body weight loss and lower replication level in FFE than SC9-2/10th in SPF chickens. However, SC9-2/40th was able to confer better immune protection as compared with CVI988/Rispens vaccination in SPF chickens. In conclusion, serially attenuation of SC9-2 in CEFs reduced the lymphoid organ atrophy and replication in SPF chickens, and the immune protective efficacy of attenuated viruses was still superior than CVI988/Rispens.

INTRODUCTION
Marek's disease virus (MDV) is one of the most contagious alphaherpesviruses and causes Marek's disease (MD), which is characterized by the development of T-cell lymphoma and the lymphocytic infiltration of peripheral nerves, skin, and visceral organs in chickens (Churchill and Biggs, 1967;Schat and Nair, 2008).
It has been demonstrated that meq-null MDV, constructed by knocking out of both copies of meq gene based on cosmid or bacterial artificial chromosome (BAC) clone of MDV, lost its tumorigenicity and provided perfect protective efficacy against challenge with MDV in chickens (Lee et al., 2008;Li et al., 2011;Su et al., 2015). However, the meq-null MDV induced lymphoid organ bursa and thymus atrophy (BTA) and body weight loss in specific pathogen-free (SPF) chickens (Lee et al., 2012). GX0101 is a field MDV strain isolated in China and has a unique genomic structure with a reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert Su et al., 2013). SC9-2 is a meq-null MDV strain constructed by knocking out both copies of meq gene and the BAC bone of GX0101 BAC clone using homologous recombination technique . Our previous studies also demonstrated that SC9-2 virus provided protection superior to CVI988/Rispens, but it induced immunosuppressive effects in SPF chickens . Published report suggests that attenuation of virulent MDV by cell culture passage on chicken kidney cells, duck embryo fibroblast, or chicken embryo fibroblast (CEF) is an effective way to decrease the viral pathogenicity (Churchill et al., 1969;Dudnikova et al., 2009;Lee et al., 2012). To eliminate the immunosuppressive effects of SC9-2, serially passages were carried out on CEF cell cultures in the present study. The pathogenesis of SC9-2/10 th and SC9-2/40 th viruses, as well as their protective efficacy against challenge with very virulent (vv) MDV rMd5, were then evaluated in SPF chickens.

Ethics Statement
The study protocol and all animal studies were approved by the Shandong Agricultural University Animal Care and Use Committee (SACUC Permission number: AVM01736-1) and performed in accordance with the "Guidelines for Experimental Animals" of the Ministry of Science and Technology (Beijing, China). Any bird deemed to have reached the humane endpoint was culled.

Cell Cultures and Viruses
Specific pathogen-free chickens and chicken embryos for preparation of CEF cells were from SPAFAS Co. (Jinan, China). Chicken embryo fibroblast cell cultures were used for virus propagation and continuous cell culture passages. The meq-null MDV strain SC9-2 was preserved in our lab and stored in liquid nitrogen . Recombinant Md5 virus (rMd5) was generated from cosmids derived from the vv MDV Md5 strain as previously described (Reddy et al., 2002). The commercial MDV vaccine CVI988/Rispens was obtained from Beijing Lingyu Company (Rispens, et al., 1972a,b).

Attenuation of SC9-2 Virus
The CEF-passed SC9-2 construct at passage 9 was serially passaged in CEF cell cultures. Marek's disease virus SC9-2 was inoculated in the CEF monolayer cell culture at a multiplicity of infection of 0.3. When plaque formation was advanced 4w5 D postinfection (D.p.i.), cells were washed with 2 mL PBS, digesting with 1 mL of 0.25% trypsin, and then resuspended with Dulbeco's modified Eagle medium supplemented with 5% fetal bovine serum. Cell containing virus were then inoculated in new monolayers of CEF for the passage of MDV SC9-2. Samples of infected cells were cryopreserved starting at 10th until 40th, and these viruses were designated as SC9-2/10 th or SC9-2/40 th . The titer of MDV was measured by counting the plaques on the CEFs. Briefly, the virus was serially diluted, inoculated on the CEFs, and incubated for 5 to 7 D at 37 C in a humidified atmosphere containing 5% CO 2 . Plaques on the CEFs were counted, and the virus titer was finally calculated.
Effect of SC9-2/10 th and SC9-2/40 th on Immune Organs At 14 D.p.i., 12 chickens per group were used to evaluate lymphoid organ BTA. The whole body weights of each chicken were measured prior to euthanasia, and all thymus lobes, bursa and spleen from each chicken were weighed after collection. The relative weights of the thymus, bursa, and spleen to the whole body were determined.
Effect of SC9-2/10 th and SC9-2/40 th on Body Weight Body weight measurements of the chickens in the different groups were performed at 7, 14, 21, and 28 D.p.i to evaluate the effect of SC9-2/10 th or SC9-2/ 40 th virus on growth rate.

Kinetics of Replication of SC9-2/10 th and SC9-2/40 th in Feather Follicle Epithelium
Feather Follicle Epithelium (FFE) samples were collected from 5 chickens of each group on 7, 14, 21, and 28 D.p.i., and DNA from FFE were prepared using standard procedures as previously described (Abdul-Careem et al., 2006). The MDV-specific primers were designed to be specific for the unique molecular marker of REV LTR in GX0101. Absolute quantification of virus copies of SC9-2/10 th or SC9-2/40 th in chickens was carried out using the real-time quantitative PCR as previously described (Su et al., 2015). The chicken ovo gene (GeneBnk NO. Y00407) was cloned into the pMD18 T vector to construct the plasmid pMD18-T-ovo. The plasmid GX0101 BAC or pMD18-T-ovo was diluted to 10 9 copies per 2 uL and serially diluted to 10 copies per 2 uL by 10 times gradient dilution. The Ct value of GX0101 BAC and pMD18-T-ovo from 10 9 to 10 1 copies were detected, and the standard curve was established on the ABI PRISM 7,500 sequence detection system. Each reaction system contains 0.5 uM MDV-specific primers, 0.2 uM LTR probe, 10 uL 2 ! TaqMan Gene Expression Master Mix buffer, and 2 uL of DNA (approximately 200 ng). The reaction volume was brought up to 20 uL by the addition of water. An ABI PRISM 7,500 sequence detection system (Applied Biosystems) was used to amplify and detect the reaction products under the following conditions: 50 C for 2 min and 95 C for 10 min, followed by 40 cycles of 94 C (15 s), and 60 C (1 min).

Antibody Responses to NDV and AIV-H9 Inactivated Vaccines
To compare the immunosuppressive effect of SC9-2/ 10 th or SC9-2/40 th virus on the antibody response to vaccinations, all chickens from each treatment were vaccinated with NDV and AIV-H9 inactivated vaccines as previously described after 6 D.p.i. (Sun et al., 2007).
On 28, 35, and 42 D postvaccination, sera from chickens of each group were randomly collected to measure hemagglutination inhibition antibody titers to NDV and AIV-H9. First, 2-fold series dilution of the collected sera were reacted with 4-hemagglutining unit virus of NDV or AIV-H9 respectively. The 1% red blood cell of chicken suspension was then added as an indicator of the hemagglutination inhibition titers.
Compare the Pathogenic Properties of SC9-2/10 th and SC9-2/40 th After inoculation with viruses, all chickens were evaluated daily for symptoms of MD and were euthanized and examined for gross lesions, when they showed clinical evidence of MD. All surviving chickens were sacrificed for necropsy to evaluate the presence of gross lesions after 13 wk of observation. Cumulative mortality and gross tumor rate were used for comparing the pathogenicity of SC9-2/10 th and SC9-2/40 th .

Experimental Design 2
To evaluate the vaccine efficacy of SC9-2/10 th , SC9-2/40 th as compared with that of the commercial CVI988/Rispens in SPF chickens when challenged with rMd5, 100 one-day-old SPF chickens were randomly divided into 4 equal groups (25 in each group) and reared separately in isolators. On day 1, 2000 PFU of SC9-2/10 th or SC9-2/40 th were inoculated i.a. into each chicken in groups 1 or 2, and chickens in group 3 received 2,000 PFU of CVI988/Rispens. No vaccine was inoculated for group 4. Five days later, each chicken in groups 1, 2, 3, and 4 was challenged i.a. with 1,000 PFU of vv MDV strain rMd5. Chickens, which inoculated with uninfected CEF cultures in the experiment 1, were the control ones as group 5. During 90 D after the challenges, all dead chickens were recorded and necropsied. At the end of the experiment, all surviving chickens were killed and necropsied. Vaccinal immunity to MD was expressed as a protective index calculated as the percentage of gross MD in nonvaccinated challenged control chickens minus the percentage of gross MD in vaccinated, challenged chickens divided by the percentage of gross MD in nonvaccinated challenged control chickens ! 100.

Statistics Analysis
Statistical analysis was performed with the GraphPad Prism (version 5.0). Differences between groups were examined for statistical significance by one-way ANOVA with Tukey's multiple comparison tests.

RESULTS
Effect of SC9-2/10 th and SC9-2/40 th on Immune Organ As shown in Table 1, the relative thymus and bursa weight of chickens in SC9-2/40 th group were significantly higher than those of chickens infected with SC9-2/10 th or GX0101 on 14 D.p.i (P , 0.05), but the relative spleen weight of chickens from the SC9-2/40 th group were significantly lower (P , 0.05). When compared with the chickens in the control group, the relative thymus and bursa weight of chickens were lower, and the relative spleen was higher in chickens of all virus inoculated group (P , 0.05).
Effect of SC9-2/10 th and SC9-2/40 th on Body Weight Compared with the uninoculated controls, GX0101 virus infection had an obvious inhibitory effect on the growth rate of the infected chickens ( Figure 1). The body weights of chickens in the SC9-2/40 th inoculation group were significantly higher than both of the SC9-2/10 th inoculation group and the GX0101 inoculation group (P , 0.05). However, the body weights of chickens infected with SC9-2/40 th were lower than the uninoculated controls (P , 0.05).
Virus Copies of SC9-2/10 th and SC9-2/40 th in FFE Virus copies in FFE were assessed with quantitative PCR at different time points postinoculation with MDVs ( Figure 2). As shown, the genome copy numbers of the SC9-2/40 th virus from FFE of infected chickens peaked at 14 D.p.i. and that of the SC9-2/10 th virus peaked at 21 D.p.i. The genome copy numbers of SC9-2/40 th virus were lower than those of the SC9-2/10 th virus (P , 0.05), except at 14 D.p.i.
Comparison of the Effect of SC9-2/10 th and SC9-2/40 th on Antibody Responses As shown in Table 2, the GX0101-infected chickens exhibited weaker humoral immune responses to the inactivated NDV and AIV vaccines as compared with those of the control ones at 28, 35, and 42 D postimmunization (P , 0.05). The SC9-2/40 th -infected chickens showed similar antibody levels as compared with those of the SC9-2/10 th inoculation ones, and both of which were higher than those of the GX0101-infected ones (P . 0.05, Table 2). However, the SC9-2/40 th -infected chickens showed lower antibody levels compared with the uninoculated controls (P , 0.05).

DISCUSSION
Marek's disease is one of the major tumor disease in poultry. Although MD has been effectively controlled by vaccines, virulence of field isolates of MDV has continued to evolve toward greater virulence and even vv plus MDV under the immune pressure of vaccines (Read et al., 2015;Ralapanawe et al., 2016). The increased virulence of field MDV led to the upgrading of vaccines from a no pathogenic herpesvirus of turkey to the attenuated CVI988/Rispens. However, it has been shown that the widely held "gold-standard" vaccine CVI988/Rispens does not confer adequate protection against MDV (Kreager, 1998;Yu et al., 2013;Zhang et al., 2015;Cui et al., 2016). Unfortunately, more than 10 strains of candidate recombinant vaccine viruses have been investigated, but none showed a better protection against MD than the CVI988/Rispens virus (Witter and Kreager, 2004).
The meq-null recombinant MDV SC9-2 was demonstrated to provide superior protection against MDV than CVI988/Rispens but induced lymphoid organ BTA and body weight loss in SPF chickens in our previous studies . Considering that attenuation of MDV is an important tool used in generating new candidate vaccines, SC9-2 virus was serially passaged in CEF cell cultures for 40 passages in the present study. Our study found that both the severity of BTA and spleen enlargement in SC9-2/40 th inoculated chickens were significantly weaker than the SC9-2/10 th inoculated ones, whereas the body weight of chickens inoculated with SC9-2/40 th were significantly higher. Chickens inoculated with SC9-2/40 th and SC9-2/10 th showed similar antibody levels induced by AIV/NDV inactivated vaccines, both of which were lower than uninoculated controls. This clearly suggests that although SC9-2/40 th virus still showed slightly immunosuppressive effect, it was much weaker as compared with the parental GX0101 in SPF chickens. The effect of attenuated virus on the antibody responses induced by Table 2. Influences of SC9-2/10 th and SC9-2/40 th viruses infection on HI antibody titers to NDV and AIV-H9 after vaccination (log2).   inactivated vaccines should also be taken into account when MDV are serially attenuated and to be used as safe vaccines. In addition, replication of SC9-2/40 th was significantly lower than SC9-2/10 th in FFE of the infected chickens. It is probably that the horizontal transmission capacity of SC9-2/40 th was weaker than SC9-2/10 th . Previous studies have shown that attenuation of MDV by serial passages decreased virus titer in vivo and reduced propensity for contact spread (Schat et al., 1985). It has been reported that the meqdeleted strain rMd5DMeq, constructed using cosmid clone technology, lost the ability to replicate in lymphocytes after serially passaged in duck embryo fibroblast cell cultures for 40 passages (Lee et al., 2012). The capacity for the virus to induce lymphoid organ BTA and body weight loss in MDV maternal antibody negative chickens were also lost. Data in their paper suggest that there is an association between viral replication in vivo and lymphoid organ BTA, which is consistent to our results. Although the SC9-2/40 th virus showed decreased lymphoid organ BTA and viral replication as compared with those of SC9-2/10 th , it still induced the lymphoid organ BTA as compared with uninoculated control in chickens. The reason for the difference between SC9-2/40 th and passage 40 of rMd5Dmeq in terms of the immunosuppressive efficacy and viral replication may be the parental virus GX0101, which is a vv virus with a high transmission capacity but weaker pathogenicity than rMd5. Our published report suggests that the REV LTR insert in the genome significantly increased the replication and horizontal transmission of GX0101 virus . The LTR deleted virus GX0101DLTR-induced slighter thymic atrophy as compared with the parental virus GX0101. Previous studies on RM1, which also contain an REV LTR, demonstrated that the recombinant virus causes more severe thymic atrophy than the parental virus JM strain, which is consistent to our studies (Witter et al., 1997). Therefore, there is also a close relationship between LTR and thymic atrophy induced by the recombinant viruses. Destruction of B and T cells account for early-MDV-immunosuppression in the lymphoid organs, resulting BTA, but not for the late-MDVimmunosuppression (Faiz et al., 2016). The mechanisms involved and the factors influencing the late-MDVimmunosuppression involving the efficacy of other vaccines are poorly understood (Faiz et al., 2017). The reason for the relative constant antibodies induced by inactivated vaccines remains to be determined.
We also evaluated the protection efficacy of SC9-2/ 40 th or SC9-2/10 th against challenge with vv rMd5 strain. Our results showed that SC9-2/40 th provided protection superior to CVI988/Rispens in SPF chickens ( Table 3). The protection index of SC9-2/40 th was slightly lower than that of SC9-2/10 th , but the difference was not significant. The results indicate that attenuation of SC9-2 virus for 40 passages on CEF has no influence on its protective efficacy. Whether the protective efficacy could be influenced after further passages needs further research. CVI988/Rispens virus has been attenuated by cell culture passage to generate a safe vaccine, which had been used worldwide for controlling MD in the field (Rispens, et al., 1972a,b). However, direct attenuation of virulent viruses by cell culture passage is indeed a long process and remains much uncertainty (Spatz et al., 2008). Our and other studies supported that deletion of major pathogenic gene in MDV using a gene knockout system combined with attenuation of MDV in cell culture may be an effective tool for generating new candidate vaccines.
In conclusion, serial passage of SC9-2 on CEF cells decreased the severity of BTA, spleen enlargement, and body weight loss caused by the virus, whereas there was no change in the protective efficacy. Passage 40 of SC9-2 provided protection superior to CVI988/Rispens in the SPF chickens. The immunosuppressive effect of SC9-2 may be eliminated by further attenuation of this virus on cell cultures to generate a safe and effective vaccine against MD.


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Yield and Profitability Analysis of Pulse and Oil Seed Based Cropping Patterns against Aman- Boro- Fallow Cropping Systems in Magura

Background: Bangladesh is one of the densely populated countries in the world. To meet up the increasing food demand there’s no alternative to increase the cropping intensity through high yielding and short duration crop varieties. Currently, cropping system of Bangladesh is mostly rice based which is also the staple food. Therefore, farmers which follow rice-rice based cropping patterns are gaining less profit day by day. Contrary, intensive rice culture is depleting soil properties gradually. Thus, to preserve soil health and increase profit pulse and oil seed based cropping patterns are crucial for sustaining a cropping pattern. This study was undertaken to find out a cropping pattern with higher yield and profit in the context of Magura district.Methods: Five cropping patterns, CP1 = Transplanted Aman rice (cv. Binadhan-7)- Mustard (cv. Binasarisha-9)- Boro rice (cv. Binadhan-14); CP2 = T.Aman (cv. Binadhan-16)-Mustard (cv. Binasarisha-10)- Boro (cv. Binadhan-14); CP3 = T.Aman (cv. Binadhan-17) -Mustard (cv. Binasarisha-10)- Boro (cv. Binadhan-14); CP4 = T.Aman (cv. Binadhan-17)- Lentil (cv. Binamasur-8)- Sesame (cv. Binatil-3) and CP5 (Control) - Aman (cv. Shorna) – Boro (cv. Heera) - fallow were assessed to identify the most suitable and profitable pattern as well as to enhance the cropping intensity by adding a pulse or oil seed crop between two rice crops. The investigation was conducted at farmer’s field of Magura during 2018-19. Block farming method was followed for experiment set up. Result: It was revealed that, maximum rice equivalent yield (REY) was obtained from CP1 (14.46 t/ha) followed by CP4 (13.52 t/ha), CP3 (13.35 t/ha), CP2 (13.09 t/ha) and CP5 (11.33 t/ha) during one year crop cycle. Highest gross margin (Tk. 1,90,189) and MBCR (1.83) was obtained from the cropping pattern CP4 and the lowest gross margin (Tk. 69,271) and MBCR (1.24) was found with the pattern CP5. Based on the above results it can be suggested that, Aman rice-Lentil-Sesame pattern i.e. CP4 may be a best choice for the farmers of Magura region for the maximum utilization of their land and gaining more profit compared to the other studied cropping patterns.

INTRODUCTION
Bangladesh is th e 8 th most populous country with a population nearing 163 million. It has a population density of 1,115.62 people per square kilometer (World Population Review, 2019). Total area of the country is 147,570 square kilometres (56,980 sq mi). Therefore, due to drought, salinity and unsuitable lands, crop production is limited to more or less 70% of the total area; which is decreasing at a rate of about 1% annually . The national average of cropping intensity is almost 2% (FAO, 2016). The opportunity for horizontal extension of cultivable land is quite impossible but cropping intensity can be enhanced to 400%. This may be achieved by adopting a series of modern technologies such as new modern high yielding short duration and stress tolerant varieties of mustard, potato, pulse, j ute and aus rice. Recently with the development of early maturing varieties of the above crops; space have been created to accommodate four crops in same piece of land in a year (BARI, 2017). To feed the future upcoming generations, gain food security and increase productivity by 50%, there is no alternative for increasing Yield and Profitability Analysis of Pulse and Oil Seed Based Cropping Patterns against Aman-Boro... cropping intensity. On the other hand, yield gap has to be reduced by eradication of monocropping to get the desired productivity of a crop variety (Islam et al., 2018a). As a result, agronomists are in challenge to develop improved crop production technologies, address production problems, processes and management of problems to maximize the production efficiency of an individual crop with optimum management practices .
Cropping pattern is the yearly sequence, temporal and partial arrangement of growing crops in a given land area. A cropping system is the crop production activity of a farm which includes all cropping patterns grown on the farm resources, other household enterprises and the physical, biological, technological and socioeconomic factors or environments (Islam et al., 2018b). Total cultivable land of Magura district is 76,701 ha. Among this, dominant cropping pattern is Boro-fallow-T. Aman; which occupying about 21,880 ha of land (28%). Pulse-Jute-fallow and Wheat-Jute-T. Aman based pattern occupies 11,033 ha (14%) and 10,618 ha (14%) of land respectively. Rest are minor cropping patterns which contributes less than 10% of the total arable land (DAE, 2019). Among the total arable land; 51,340 ha (BARC, 2020a) and 9,463 ha (BARC, 2020b) is very suitable for production of lentil and mustard respectively.
Rice is the staple food of Bangladesh and it is extensively produced over 70% of its total agricultural lands (Chowhan et al., 2019). Intensive rice-rice cropping systems results in lessening of soil pH and thereby creating soil acidity problem which ultimately results in unavailability of nutrients, depletion of soil and lowering crop yields. Crop rotation with pulse or oil seeds can be easily included in between two rice crops to sustain productivity and profitability of rice based production system (Singh et al., 2019). Sequential cultivation of different crop ensures utilization of all set of nutrients within the soil. It also contributes to reduce soil erosion, enhance soil fertility and crop yield (Adarsh et al., 2019). Cost of rice production is being rising every year; whereas, market price is not up to the mark. Thus, making it a less profitable crop. Production cost of pulse and oil seed crops is much less compared to rice. Conversely, profit is more than rice. Keeping the above factors in mind the present research program was aimed to select suitable crop(s) and pattern to raise yield and cropping intensity and to find out the most profitable cropping pattern in the context of Magura.

MATERIALS AND METHODS
The study was conducted at the farmer's field of Maghi under the sadar upazila of Magura district during July 2018 to June 2019. Geographical position of the experiment site was 2325'51.7"N latitude and 8923'54.1"E longitude. It was under the AEZ (Agro Ecological Zone) 11; which is high ganges river flood plain and mostly high to medium high land type characterized by lower content of organic matter and fertility level (Table 1). Total area of AEZ 11 is 1320549 ha under this Magura holds 85700 ha of land (Das et al., 2018;Ahmed et al., 2018). It belonged to tropical monsoon climate with unimodal rainfall. Average weather data  of Magura district is presented in Fig 1 (BINA, 2019a). Block farming approach was followed for experiment set up. The following five cropping patterns (CP) were laid for the experimentation- Land area for each pattern (block) was 0.25 acre (25 decimal) and total area was 1.25 acres for all the five patterns. For rice, land preparation, planting method, time, seedling age, weeding, pest control, rouging etc. were done according to the methodology followed by Chowhan et al. (2017) and Chowhan et al. (2019). In case of mustard, seeds were broadcasted at the rate of 3.0 Kg/ha. They were thinned and weeded after 15-20 days to maintain plant population of 50-60/m 2 . To prevent from Alternaria blight and other fungal diseases Rovral-50 W P and Autostin-50 WDG were sprayed @ 3g and 2g/L respectively. Insects were controlled by applying Reeva 2.5 EC @ 2.5ml/L when necessary. Seeds were harvested by cutting the crop plants from ground level when siliquae attained a maturity of 80%. For lentil, seeds were treated with Provax 200 W P @ 3g/kg seed and broadcasted @ 15 kg/acre. Weeding, thinning and mulching were done at 30 DAS (days after sowing). For effective controlling of foot and root rot, Provax 200 WP was sprayed @ 2.5g/L thoroughly with care into the root zone at 12 days interval for 3 times. Ripcord 10 EC was applied @ 2ml/L for controlling insects. During early maturity period Amistar Top 325 EC was sprayed 2 times at 10 days interval @ 3ml/L. Tilt 250 EC @ 2.5ml/L was used to prevent rust of lentil. When the pods matured and the color turned dark yellow to brown whole plants were harvested and the seeds were separated from the pods by a mechanical thresher. Sesame seeds were sown @ 3.25kg/acre by broadcasting. Land preparation of sesame was done according to Paul et al. (2019). Plots were kept weed free up to 25 days. Insect infestation was checked through Karate 2.5 EC @ 3ml/L. Fungal diseases were stifled by Forastin 50 WP @ 2.5g/L. Plants were harvested by cutting when plant and capsules turned yellowish color from the bottom. Then the collected plants were sun dried evenly up to complete drying. Later seeds were extracted manually by labor. A well drainage facility was ensured in all the patterns to prevent excess moisture stress. Fertilizer dose (N-P-K-S-Zn-Mg-B kg/ha) for Aman rice was 60-10-50-8-0.8-0-0, Mustard was 80-24-60-18-1.5-3.0-1.0, Boro rice was 120-16-76-12-1.3-0-0, lentil was 80-24-60-18-1.5-3.0-1.0 and sesame were 120-16-76-12-1.3-0-0. Fertilizers were applied as per crop and time stated in FRG (2012). After the end of each crop's harvest, data were Yield and Profitability Analysis of Pulse and Oil Seed Based Cropping Patterns against Aman-Boro... collected on yield and yield attributes, gross return, total variable cost, gross margin and BCR (Benefit cost Ratio). Then, summation of different parameters of the individual crop's result was done separately and average performance was calculated.
Rice Equivalent Yield (REY) was used to compare between crop sequences, the yield of all crops was converted into rice equivalent on the basis of prevailing market prices of individual crop (Verma and Modgal, 1983). Following formula (Kamrozzaman et al., 2016) was employed to determine REY-REY (t/ha) = Economic analysis included data collection on prices and quantities of inputs used and output produced (seed, straw, byproducts etc.). The inputs used included seed, fertilizer, labour and pesticides. The output and inputs were valued at market prices. Results were used to count net income, benefit cost ratio and rice equivalent yield of crops. Net income was computed as the difference between management (family labour and operator's) cost and gross margin. BCR was computed Kamrozzaman et. al. (2016) as per the following formula:

Crop duration and turnaround time of patterns
In the first cropping pattern (CP 1 ) Binadhan-7 was transplanted within 25 DAS; which was harvested at field duration of 93 DAT (days after transplanting) (  First crop (Binadhan-17) of CP 3 was transplanted at 20 DAS. Field maturity of Binadhan-17 was 97 days (Table 2). Average duration of 30 demonstrations of Binadhan-17 at Magura was found to be 116 days by BINA (2019b). The next crop in this pattern, mustard (Binasarisha-10) had a duration of 83 days. After harvesting mustard, there were 28 days turnaround time for transplanting Binadhan-14. 28 days old seedlings were transplanted in the main field. Binadhan-14 matured within 90 DAT. Less duration may have occurred due to soil and weather conditions. Fourth cropping pattern's (CP 4 ) first crop aman rice, Binadhan-17 was transferred to main plot at 27 DAS. It gained maturity within 92 days (Table 2); later 13 days turn around time was obtained for sowing lentil (Binamasur-8).
Harvesting of Binamasur-8 was done 106 DAS. Earlier studies (BINA, 2019b) mentioned that, mean duration of Binamasur-8 in Magura was 104 days. Which was close to the present findings. Sesame was sown after 11 days of harvesting lentil. Binatil-3 attained maturity 91 DAS. Investigations from Ali, (2018) and BINA, (2019b) indicated that, mean duration of Binatil-3 in Magura ranged from 81 to 88 days. W hich was in conformity with the present outcome.
CP 5 was the control pattern in the study. A wide locally cultivated aman rice (Shorna) was the first crop in this pattern. 30 days old seedlings were used for transplanting of shorna. This variety gained maturity at 99 DAT and harvested (Table 3). Rahman and Islam, (2019) observed field duration of shorna between 103 to 108 days. After aman rice the land under this pattern (CP 5 ) was kept fallow as farmers local practice. But there's a huge scope to cultivate short duration mustard varieties through zero tillage in this type of land to meet up national oil demand (TBS, 2019). In the third sequence, locally produced hybrid variety of boro rice was transplanted at 45 DAS. Heera was harvested at 100 DAT. Chowhan et al. (2019) stated that Heera-1 had a mean duration of 147.78 days with varietal effect. W hich closely supported the present findings.

Productivity and yield of five patterns
Binadhan-7 in CP 1 (Fig 2) produced a grain and straw yield of 5.3 t/ha and 5.88 t/ha respectively. This field results were in supporting with the findings of Rahman and Islam (2019); who found grain yield, 4.18 t/ha to 5.26 t/ha and straw yield, 4.85 t/ha to 5.15 t/ha on several cropping pattern experiments. Next crop, Binasarisha-9 gave 1.6 t/ha seed Bd -Binadhan, Bs -Binasarisha, Bm -Binamasur, Bt -Binatil.
In this regards you may please reply of the reviewer comments mail.
Yield and Profitability Analysis of Pulse and Oil Seed Based Cropping Patterns against Aman-Boro... yield and 3.04 t/ha straw yield. BINA (2019b) reported that mean seed yield of Binasarisha-9 demonstrations at Magura was 1.33 t/ha. Higher yield in pattern was may be due to improved management practices.  concluded that, straw yield of Bina mustard varieties were 3.74 t/ha to 4.05 t/ha. The low yield of straw may have obtained due to soil moisture, fertility and weather related factors. Last crop of CP 1 ; Binadhan-14 yielded 6.5 t/ha of grain and 6.89 t/ ha straw; this was the maximum yield obtained among the three cropping patterns (CP 1 -CP 3 ). Similar results on yield were mentioned by BINA (2019b). First crop of CP 2 (Fig 2), Binadhan-16 attained a grain and straw yield of 4.61 t/ha and 5.24 t/ha. These results are alike with the findings of Chowhan et al. (2019). Binasarisha-10, the second crop of this pattern gave less production than CP 3 . It obtained 1.3 t/ha seed yield and 2.14 t/ha straw yield. Ahmed and Kashem (2017) achieved similar outcomes with BARI Sarisha-14. Finally, third crop Binadhan-14 gave 6.31 t/ha grain yield and 6.72 t/ha straw yield; which was the second highest yield compared to the first three patterns. This finding was in affirmatively supported by Chowhan et al. (2019).
In CP 3 (Fig 2), Binadhan-17 yielded more than CP 4 . It gave 5.15 t/ha grain yield and 6.15 t/ha straw yield. Chowhan et al. (2017) reported 5.05 t/ha and 7.58 t/ha grain and straw yield of Binadhan-17 in aman season. Differences in straw yield might have occurred for fertilizer management and crop duration. A seed and straw yield of 1.44 t/ha and 2.89 t/ha was given by Binasarisha-10 which was slightly more than CP 2 's mustard yield. Conversely, Binadhan-14 produced 5.8 t/ha and 6.27 t/ha grain and straw yield, which was lowest among the first three patterns (CP 1 -CP 3 ).
Fourth pattern's (CP 4 ) first crop (Fig 3), Binadhan-17 gave lesser production compared to CP 3 (Fig 3). 4.88 t/ ha grain and 6.12 t/ha straw yield were gathered from Binadhan-17 in CP 4. The next crop, lentil (Binamasur-8) gave a seed and straw yield of 1.87 t/ha and 2.02 t/ha serially. Haque et al. (2015) found varietal impact of different BARI Masur gave seed yield between 0.94 t/ha to 1.28 t/ha and straw yield of 2.2 t/ha to 2.48 t/ha. BINA (2019b) reported that, mean yield of Binamasur-8 from field demonstrations of Magura were 1.812 t/ha. These consequences were consistent with the present outcomes. Binatil-3 was the final crop in CP 4 . It provided a seed and straw yield of 1.26 t/ha and 2.32 t/ha. BINA (2019b) stated mean seed yield of Binatil-3 in farmers' field demonstrations were 1.16 t/ha. Which validates the current finding.
Control pattern (CP 5 ) had only two crops in the cropping sequence i.e. aman and boro rice (Fig 3). First crop, Shorna gave 4.25 t/ha of grain and 5.56 t/ha of straw yield. Rahman and Islam (2019) concluded, grain yield extent of Shorna was 4.22 t/ha to 4.80 t/ha and straw yield was 4.40 t/ha to 5.10 t/ha. W hich was consistent with the present result.   Heera gained a yield of 7.08 t/ha in grain and 10.14 t/ha in straw. Tiongco and Hossain, (2015) reported that, Heera is a very popular grown dry season (boro) rice and its mean grain yield was 7.632 t/ha. Variation of yield in Heera rice in the pattern (CP 5 ) may have happened due to cultural, environmental and fertilizer management practices. Within the studied cropping patterns (CP 1 -CP 5 ) highest rice equivalent yield (REY) was observed (Table 4) with CP 1 (14.46 t/ha) followed by CP 4 (13.52 t/ha) and CP 3 (13.52 t/ ha). Pattern (CP 4 ) under control (farmers practice) produced the lowest REY (11.33 t/ha). Greater REY is not always an indication of improved or profitable cropping pattern; rather it depends on the type, market value and crop sequence.

Farmers' opinion
Farmers opined that a pulse or oil crop could be grown successfully after cutting aman rice. Some of them were interested to cultivate mustard through zero tillage. Finally, they realized the importance growing pulse and oil crops for higher economic benefits. The also said, Mustard, lentil and sesame being a field crop and requirement of less input cost it will be profitable. They widely cultivate Shorna for its taste, flavor and market price and Heera for higher yield.

CONCLUSION AND RECOMMENDATIONS
From the results it can be concluded that, for magura region CP 4 =T.Aman (cv. Binadhan-17)-Lentil (cv. Binamasur-8)-Sesame (cv. Binatil-3) may be a suitable cropping sequence over the existing Aman rice -Boro rice -fallow cropping pattern. Contrary, the lag phase between sesame and aman rice in CP 4 can be exploited for cultivation of short duration vegetables. As CP 4 incorporates all the modern high yielding and short duration varieties. By adopting this pattern farmers of this region will gain higher yield, profit and ensure the maximum utilization of land and time. Though this research explored only a limited area of Magura sadar upazila; further verification and investigation is required for more authentication of the above outcomes.