extfieldsofstudy: Medicine Biology

text:
Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system

Summary Huanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium, Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB‐tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB‐tolerant citrus hybrid (Poncirus trifoliata × Citrus reticulata) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB‐tolerance. These regulators display similar expression patterns in another HLB‐tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid (Microcitrus virgata). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus‐induced gene silencing in N. benthamiana, this innovative and cost‐effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1‐Associated Protein, and the negative regulator Vascular Associated Death Protein.

Introduction
Huanglongbing (HLB), also known as citrus greening disease, is the most destructive disease threatening the global citrus industry. Since the first US case of HLB was discovered in Florida in 2005 (Stokstad, 2012), the disease has spread to most citrus production states, including Texas and California. Despite extensive efforts and research, there are no effective control strategies for the disease. HLB is caused by a fastidious bacterium Candidatus Liberibacter asiaticus (CLas), which is transmitted by the insect vector Asian citrus psyllid (ACP) Diaphorina citri (Bove, 2006). Along with drastic losses in fruit production, the need to increase insecticide applications for vector control has raised costs significantly, making citrus production for growers very difficult in affected areas. The current lack of methods to effectively combat HLB makes discovery of innovative control strategies imperative for the survival of the citrus industry.
Almost all citrus species including most commercial varieties are susceptible to CLas, with the exception of a few tolerant citrus relatives, such as Poncirus trifoliata L. Raf. and some of its hybrids Bowman, 2011, 2012a), which are commonly used as rootstocks. These HLB-tolerant varieties are a valuable resource for the discovery of the natural important regulators involved in defence against HLB. US-942, a hybrid of Sunki mandarin (Citrus reticulata) and trifoliate orange (Poncirus trifoliata), is one of the most popular HLB-tolerant rootstocks in Florida because it can induce production of good yield and fruit quality of commercial scions under HLB-endemic conditions . However, the functional regulators linked to the HLB-tolerant phenotype of US-942 are still unclear (Albrecht and Bowman, 2012a,b;Albrecht et al., 2016).
Plant endogenous small RNAs (sRNAs), including both micro-RNAs (miRNAs) and small interfering RNAs (siRNAs), have emerged as important regulators of gene expression in plant immune responses against pathogen infection (Cai et al., 2018;Huang et al., 2019;Li et al., 2017). Bacterial infection can lead to up-or down-regulation of sRNAs, which then suppress or desuppress expression of their target genes, contributing to plant defence (Katiyar-Agarwal et al., 2007;Katiyar-Agarwal et al., 2006;Navarro et al., 2006;Zhang et al., 2011). sRNA-mediated regulatory mechanisms have also been identified in crop plants by characterizing host sRNA profiles during pathogen attack (Huang et al., 2019). In the citrus-CLas pathosystem specifically, a high accumulation of the miR399 in CLas-infected trees revealed a link between phosphate starvation and HLB response (Zhao et al., 2013). Here, we report that sRNA profiling is a powerful tool for identifying key regulators of citrus defence responses against CLas. Through comparative analysis of sRNA profiles of the HLBtolerant hybrid, US-942 and the HLB-susceptible Cleopatra mandarin, we have identified panels of sRNAs and their targets with unique response patterns to CLas in the US-942 background and revealed potential important regulators contributing to the HLB-tolerant phenotype. The expression patterns of these candidate regulators in the tolerant US-942 background are similar to those in the genetically distinct and geographically separated HLB-tolerant citrus relative, Sydney hybrid (Microcitrus virgata), further supporting that the genes identified in the current study are important regulators associated with HLB-tolerance.
Thus far, functional studies of HLB-tolerance related genes have been hampered by the inability to obtain a pure culture of CLas, the significant time and effort required for genetic study of gene functions in citrus trees, and the vector-mediated transmission nature of the pathogen. A rapid large-scale functional screening method for host immune regulators that contribute to defence against C. Liberibacter species is urgently needed. To fill this need, we developed an effective host/vector/pathogen interaction system using a close relative of CLas, C. Liberibacter solanacearum (CLso), which infects solanaceous plants, the potato psyllid, a major pest of potatoes and tomatoes, and Nicotiana benthamiana, the ideal hosts for virus-induced gene silencing (VIGS) experiments. VIGS is an effective silencing method to knock down expression of plant endogenous genes using a viral vector. This system is very similar to the natural citrus/ psyllid/CLas interaction system and can be used to rapidly characterize the function of candidate regulators in plant defence responses against C. Liberibacter species.

Results
A set of miRNAs and siRNAs is more abundant in the tolerant hybrid US-942 Through comparing the sRNA profiles of uninfected HLB-tolerant hybrid US-942 and uninfected HLB-sensitive mandarin Cleopatra, we discovered 19 conserved miRNAs which are constitutively more abundant in US-942 than in the HLB-susceptible Cleopatra when using >5 reads per million total normalized reads (rpm) and >3 fold change as cutoffs (Table S1). Three miRNAs are constitutively less abundant in US-942 than in Cleopatra (Table S1). MiR399 has a much higher constitutive expression level in US-942 than in Cleopatra and is further induced upon CLas infection (Figure 1a). Our previous study discovered that miR399 expression is highly induced after CLas infection in most HLB-susceptible citrus varieties (Zhao et al., 2013). In Arabidopsis, miR399 expression is induced by P i -deficiency and plays a role in the regulation of P i homoeostasis (Hsieh et al., 2009). Previously, we demonstrated that HLB-positive citrus trees contain significantly lower levels of phosphorus (P i ) than uninfected trees and reported this as one of the major causes for HLB disease symptoms (Zhao et al., 2013). MiR399 targets and silences an E2 ubiquitin conjugating enzyme (PHO2) that is responsible for the degradation of P i transporters, which results in increased P i transporters and P i uptake (Zhao et al., 2013). Here, we show that the target gene of miR399, PHO2, has significantly lower expression in US-942 compared to Cleopatra with or without CLas infection (Figure 1b,c). Additionally, the two P i transporters, PT2 and PHT2, both have higher expression levels in US-942 than in Cleopatra (Figure 1b,c). Thus, gene regulation mediated by enhanced expression of miR399 makes US-942 better equipped to cope with P i starvation after CLas infection.
To probe whether siRNAs of US-942 are also expressed differentially from Cleopatra, we first focused on siRNAs which have differential constitutive expression in US-942 in comparison to Cleopatra. To do this, we compared the siRNA profiles of uninfected US-942 and Cleopatra and identified a panel of siRNAs with 3-fold higher or lower expression in US-942 than in Cleopatra. 209 siRNAs have higher constitutive expression levels in US-942, which remain high upon CLas infection (Table S2). In order to determine what proportion of these sRNAs were contributed to US-942 by the P. trifoliata genome, we searched these 209 sRNA sequences in the P. trifoliata genome database. We found that 49% of these sRNAs (103 out of 209 sRNAs) could be encoded by P. trifoliata genome (Table S2). In silico target prediction of identified siRNAs was performed against the transcript database of Citrus clementina, the closest relative to US-942 with an available sequenced genome, using the base pairing prediction score ≤4 as a cutoff (Dai et al., 2018). The target genes of these siRNAs would be silenced to a greater degree in US-942 than in Cleopatra and could play a negative role in natural defence against HLB (Table S3). Further, we identified 293 sRNAs with 3-fold lower expression in US-942 than in Cleopatra, which remain low after CLas infection (Table S4). Among these 293 sRNAs, 48%, 140 sRNAs, are represented in the P. trifoliata genome (Table S4). It is likely that these siRNAs silence target genes which contribute positively to plant defence responses (Table S5). Indeed, a Gene Ontology (GO) enrichment analysis demonstrated that the predicted target genes are enriched in GO terms relating to defence response to bacterium and callose deposition (da Huang et al., 2009) (Figure S1a). Lower expression levels of these siRNAs would lead to higher expression of these putative positive regulators, making US-942 better equipped to defend against HLB.

Identification of important defence regulators from US-942
To identify natural plant defence regulators in response to CLas infection, we focused on the sRNAs which displayed differential expression patterns both before and after CLas infection only in the HLB-tolerant hybrid US-942, because these sRNAs and their target genes likely contribute to HLB-tolerance. Regulators with both a rapid (within two months post CLas infection) and extended response (six months post CLas infection) are more likely to provide strong and durable protection from HLB than genes with transient regulatory patterns. Leaf samples from US-942 and Cleopatra at 7 and 28 weeks post grafting inoculation (wpi) of CLas were subjected to sRNA profiling, with uninfected leaves used as controls. The sRNAs with similar response patterns in both US-942 and Cleopatra were eliminated from analysis. A total of 371 sRNAs were up-regulated more than 3-fold at both 7 and 28 wpi in US-942, but not in Cleopatra (Table S6), and 100 sRNAs were down-regulated by more than 3-fold only in US-942 (Table S7). Among the 371 up-regulated sRNAs and the 100 down-regulated sRNAs, 30% and 11% were present in the P. trifoliata genome, respectively (Table S6 and S7). We selected the top fifty sRNAs from both lists and performed in silico target prediction analysis using a stringent base pairing prediction score of ≤4 as a cutoff (Table S8 and S9). These sRNA targets were enriched in the GO terms of plant defence responses, induced systemic resistance response, and hypersensitive response (Figure S1b). These genes have a high likelihood of contributing to HLB-tolerance for US-942. The target genes of the down-regulated sRNAs are potentially upregulated at the transcriptional or translational level and act as positive regulators of host defence responses against HLB (Table S9). Conversely, the target genes of upregulated sRNAs are likely to be negative regulators of plant defence genes responding to CLas infection, which are suppressed by these sRNAs (Table S8).
Candidate defence regulators identified from US-942 show similar expression patterns in another HLBtolerant citrus relative To identify potential regulators that contribute to HLB tolerance, we selected the sRNA target genes with known or predicted functions in plant defence responses for further functional analysis ( Figure 2a). The expression level of the sRNAs and their target genes in CLas-infected US-942 and Cleopatra was validated by quantitative RT-PCR (Figure 2b-e, S3 and S4). Two sRNAs specifically down-regulated in US-942 after CLas infection, 942si1001 and 942si1020 (Table S7), were experimentally confirmed to have lower expression levels in US-942. The two sRNAs specifically up-regulated in US-942 after CLas infection, 942si2003 and 942si2048 (Table S6), were confirmed to have a higher expression level in US-942 ( Figure 2b). In all four cases, target gene expression is clearly negatively correlated with the expression of corresponding sRNAs. The targets of 942si1001 and 942si1020, BRCA1-Associated Protein (BRAP) and cytochrome P450 CYP93, respectively, had 3-4 fold higher transcription levels in US-942 than in Cleopatra ( Figure 2d). BRAP is an E3-ligase which positively regulates pathogen-associated molecular pattern triggered defence response in plants and CYP93s are involved in the synthesis of a phytoalexin in soybean (Park et al., 2016;Schopfer et al., 1998). It is likely that both BRAP and CYP93 positively regulate plant defence and are upregulated in response to the down-regulation of their corresponding siRNAs during CLas infection in US-942. The targets of 942si2003 and 942si2048, Oligopeptide Transporter 1 (OPT1) and Vascular Associated Death (VAD), respectively, displayed expected negative expression correlation to their corresponding sRNAs (Figure 2b,d). In Arabidopsis, the oligopeptide transporter family mediates the uptake of a virulence factor, Syringolin A, which is secreted by plant pathogen Pseudomonas syringae pv. syringae (Hofstetter et al., 2013). Further, the vad1 Arabidopsis plant is resistant to bacterial pathogens due to increased salicylic acid accumulation (Lorrain et al., 2004). Thus, citrus OPT1 and VAD1 likely act as negative regulators of plant defence, which are silenced by sRNAs after CLas infection in US-942.
We also examined the expression patterns of these selected candidate regulators in an HLB-tolerant citrus relative, Sydney hybrid (Microcitrus virgata or Citrus virgata), a hybrid of the Australian round lime, Microcitrus australis and the Australian finger lime, M. australasica (Reuther, et al., 1967). The progenies of the Sydney hybrid display segregation of the HLB-tolerance trait (Ramadugu et al., 2016). Among them, Sydney72 (Syd72) shows strong tolerance to HLB, whereas Syd74 is highly susceptible to CLas infection. Similar to US-942, Syd72 has higher expression levels of miR399 compared to Syd74 ( Figure S2). We further demonstrate that these identified sRNAs (Figure 2b,c) and their target regulators (Figure 2d,e) displayed similar expression patterns in both HLB-tolerant hybrids, Syd72 and US-942. These results further support that these selected plant defence regulators are possibly involved in HLB-tolerance responses because they display the same unique expression pattern in both HLBtolerant hybrids. Establishment of a new Nb/Potato psyllid/CLso pathosystem that is similar to the citrus/Asian citrus psyllid/CLas pathosystem Functional characterization of genes, protectants or therapeutic treatments on citrus has been difficult due to the extended lifespan of citrus trees and the fluctuating patterns of HLB development within a tree (Fletcher et al., 2018). Further, genetic manipulation of citrus requires significant time and effort, which makes large-scale functional genetic screening almost impossible.
To validate and rapidly screen the functions of potential regulators, identified through sRNA profiling and gene expression analysis, in HLB defence response, we developed a novel screening system by infecting the model plant, Nicotiana benthamiana (Nb), with CLso through transmission by the potato psyllid. This pathosystem can serve as a surrogate for the citrus/ ACP/CLas pathosystem and allow rapid large-scale functional screening of candidate genes and other molecules. We chose to use Nb because it is an ideal host for efficient virus-induced gene silencing (VIGS; Ratcliff et al., 2001), which allows for rapid determination of gene function by knocking down gene expression using a viral expression vector. Although Nb is not a natural host for the potato psyllid and CLso, we discovered that potato psyllid nymphs can feed on very young Nb plants. Two-week-old Nb were exposed to CLso-positive potato psyllid nymphs for 5 days (Figure 3a). Three weeks later, there was observable yellowing symptoms and vascular tissue greening in leaves (Figure 3b,c). Additionally, the CLso 16S ribosomal RNA gene was detected by PCR (Figure 3d) in leaves that were directly fed upon as well as those systemic leaves. During feeding, psyllid nymphs and possibly adults, inject toxic saliva into plant foliage and cause feeding damage known as 'psyllid yellows' (Liu and Trumble, 2004). The 'psyllid yellow' symptom was observed on Nb plants exposed to both CLso-positive and CLso-negative psyllids 5 days after feeding ( Figure S5). Nb plants fed to CLsonegative psyllids recovered from the damage caused by psyllid feeding, whereas plants fed to CLso-positive psyllids developed further chlorosis symptoms (Figure 3e and S5). The CLso bacterial titre was detected by probe-based qPCR. At three wpi, the bacterial titre ranged from 10 5 to 10 8 copies per 50 ng plant genomic DNA whereas no bacteria were detected in plants exposed to CLso-negative psyllids (Figure 3f).
This model system allows us to detect CLso infection as early as three wpi. This is a significant advantage over the current system where six to 12 months may be required from the inoculation of CLas to the detection of bacteria and the development of disease symptoms in citrus (Fletcher et al., 2018). The Nb/Potato psyllid/ CLso pathosystem we report here can provide results in 6-8 weeks, saving both time and resources.
Using VIGS and the Nb/Potato psyllid/CLso pathosystem for rapid functional screening of potential regulators A key advantage of the model Nb/psyllid/CLso pathosystem, in addition to shortening disease development time, is that the expression of target genes can be easily manipulated in Nb plants by VIGS (Ratcliff et al., 2001). Tobacco rattle virus (TRV) is the most widely used VIGS vector for gene functional analysis. TRVbased VIGS vectors have been used to silence genes in a number of Solanaceous plant species and are most effective in Nb (Liu et al., 2002;Ratcliff et al., 2001). To study the function of candidate regulators in natural defence responses against CLso, we used TRV-based VIGS to knock-down the Nb orthologous/ homologous genes of the candidate citrus regulators in Nb plants infected with CLso.
The two-week-old Nb plants were exposed to CLso-positive potato psyllid nymphs for 5 days. Three days after psyllid nymph removal, during which time the plants recovered from psyllid yellows, we performed TRV-based VIGS through Agrobacterium tumefaciens mediated leaf infiltration (Lu et al., 2003). The TRV construct containing a piece of Solanum bulbocastanum-specific late-blight resistance gene, RB (Song et al., 2003), was used as a negative control (siRB), as it has no target gene in Nb. As expected, CLso disease formation and the gene knock-down effect both occurred within 2-3 weeks.
The screening results of the positive regulator, BRAP, and the negative regulator, VAD are shown in Figures 4 and 5. Compared to the negative control, knocking down BRAP led to a CLso susceptible phenotype, as indicated by stunted growth and chlorosis (Figure 4a,b), supporting that BRAP acts as a positive regulator of plant defence responses against HLB. On the contrary, knocking down the negative regulator VAD gene, revealed a CLso tolerant phenotype with weaker symptoms and, larger and healthier plants compared to the negative control (Figure 5a,b). Silencing efficiency of BRAP and VAD in all tested leaves was monitored by qRT-PCR (Figures 4c and 5c) and compared to the siRB control plants. The bacterial titre in the midvein from the leaves used to monitor BRAP and VAD silencing levels was detected by probe-based qPCR. Compared to the siRB controls, a significantly higher bacterial titre was detected in siBRAP plants ( Figure 4d) and a significantly lower bacterial titre was detected in the siVAD plants ( Figure 5d). Thus, BRAP was confirmed to be a positive regulator in plant defence response to CLso, and VAD a negative regulator.

Discussion
Growing disease-resistant or -tolerant varieties is the most ecofriendly and cost-effective strategy for disease management. In the absence of naturally resistant varieties, one of the most attractive long-term and sustainable means of disease control is to identify important regulatory molecules and regulatory pathways in disease-tolerant/resistant hybrids or closely related species, and to work in concert with these natural plant responses to improve plant productivity in an HLB-endemic environment. Currently, US-942 is one of the most popular HLB-tolerant rootstocks in Florida (Bowman and Joubert, 2020;Bowman et al., 2016;Castle, 2010) and can serve as a rich genetic source for identification of plant genes that play roles in HLB defence. Rootstocks conduct nutrients, phytohormones, sRNAs, small proteins and metabolites to the scion. Some are systemic signalling molecules and can directly or indirectly affect scion gene expression (Wang et al., 2017). Citrus rootstocks affect the scion in many other ways, including tree vigour, fruit size, fruit quality and yield (Benjamin et al., 2013;Castle, 2010). They also play an important role in plant tolerance to abiotic and biotic stress (Castle, 2010;Dutra de Souza et al., 2017;Laino et al., 2016). The use of a CLas resistant rootstock may impart a positive effect on the phloem microenvironment, possibly mitigating the negative effects of CLas on the scion. However, no reliable breeding markers for the selection of HLB-tolerant progeny are currently known. Molecular markers will significantly accelerate the identification of disease-resistant/tolerant varieties for breeding programs. The sRNAs and their target genes we identified as important regulators in this study are potentially linked to the HLB resistance/tolerance phenotype. These regulators can be developed as selection molecular markers for rootstock and scion breeding.
Comparing the sRNA profiles of HLB-susceptible and tolerant plants allowed us to reveal how the pathogen differentially affected the sRNA regulatory network in plants. From our sRNA profile analysis, it appears that US-942 has a stronger defence response to CLas infection. For example, it is likely that US-942 can take up P i more efficiently due to the high expression of miR399, contributing to the HLB-tolerant phenotype, and potentially rendering the grafted tree more tolerant to nutrient depletion due to CLas infection. Of the other miRNAs more abundant in US-942 (Table S1), miR159 and miR166 are involved in multiple hormone pathways and trigger stronger anti-bacterial defence responses (Zhang et al., 2011). MiR159, miR167, miR396 and miR408 have been demonstrated to contribute to droughtresistance (Hajyzadeh et al., 2015;Reyes and Chua, 2007;Sunkar and Zhu, 2004), and miR166, miR396, miR408 and miR477 were shown to be responsive to cold stress (Lu and Huang, 2008;Zhou et al., 2008). Taken together, these clues from differentially expressed miRNAs suggest that US-942 has a higher tolerance for certain biotic and abiotic stresses and can more efficiently take up nutrients than Cleopatra. Our results reveal that the HLBtolerance of US-942 may not only rely on a particular pathway or a single regulator, but a sum of regulators, which when combined lead to a stronger defence response, more efficient nutrient uptake and the enrichment of metabolic products that benefit the host or impair pathogen survival. The application of the regulators requires further molecular genetic approaches and functional analysis to confirm the HLB-tolerant phenotype.
The complete genome sequence of CLas shows that the pathogen is likely to be parasitic rather than pathogenic, with the disease symptoms more similar to nutrient depletion or metabolic disorder (Duan et al., 2009). Studies of the transcriptome, the proteome and the metabolome have revealed some potentially important pathways and responses that appear to play a role in host tolerance to HLB (Albrecht and Bowman, 2012b;Albrecht et al., 2016;Albrecht et al., 2020;Hung and Wang, 2018 , 2016). In our sRNA profiling study, the results of a GO enrichment analysis also indicate that genes involved in metabolic pathways, such as flavonoid biosynthesis, are regulated by sRNAs and are differentially expressed in US-942, which may also contribute to plant defence.
One of the key follow-up steps for identification of important regulators of plant defence responses against HLB is to conduct functional screening to validate and characterize the candidate regulators identified from the comparative genomic studies. Functional genetic studies on tree crops, such as citrus, require significant time and effort due to the extended lifespan of a tree, low transformation efficiency and limited genetic resources (Fletcher et al., 2018). A rapid functional screening method has been lacking for genetic analysis of important genes identified from various genomic studies, including RNA-seq, sRNA-seq, MS/ MS analysis. Recently, it was shown that Nb can be infected by CLas via transmission by the parasitic plant, dodder or Cuscuta (Pitino et al., 2018). Nb is considered the best plant host for genetic studies using VIGS to knock-down the gene of interest. CLso, which is vectored by the potato psyllid, can also infect Nb plants, but only through insect vector transmission. Although Nb is not an ideal host for potato psyllids, we identified the ideal conditions for the potato psyllid to infest Nb, which made it possible to utilize the Nb/Potato psyllid/CLso pathosystem as a model of the highly similar citrus/Asian Citrus psyllid/CLas pathosystem, for rapid functional screening of candidate defence regulators and pilot testing of new therapeutic compounds. Using this system, combined with gene knock-down studies in Nb using VIGS, we can study gene function and substantially reduce the time and cost involved in functional screening efforts. Subsequently, introducing the confirmed master regulators derived from disease-tolerant/resistant varieties, silencing the negative regulators, or gene editing the commercially important varieties will help generate HLB-resistant or tolerant commercial cultivars and reduce the chance of genetic incompatibility. This approach allows us to discover with high confidence the potential master regulators that contribute to HLB tolerance and can assist in the development of new disease control therapies.

Plant material and CLas inoculation (US-942 and Cleopatra)
One-year-old greenhouse-grown Cleopatra mandarin (Citrus reticulata) and US-942 (C. reticulata x Poncirus trifoliata) seedlings  were used. Cleopatra is susceptible to CLas, while US-942 is tolerant (Albrecht and Bowman, 2012a). Plants were infected with CLas through grafting with buds from infected greenhousegrown citrus plants. Control plants were mock-inoculated with buds from healthy disease-free citrus plants. Plants were cut back immediately after inoculation. For CLas detection DNA was extracted from 100 mg of ground leaf tissue using the Plant DNeasy Mini Kit (Qiagen) and real-time PCR assays were performed using primers HLBas and HLBr and probe HLBp (Li et al., 2006). For sRNA library construction, three healthy plants (control) and three CLas-infected plants of Cleopatra and US-942 with similar patterns of disease progression were used. Leaves and stems of plants were harvested 7 and 28 weeks post inoculation (wpi), immediately frozen in liquid nitrogen, and ground in liquid nitrogen using mortar and pestle. CLas inoculated Cleopatra plants were reduced in growth and displayed leaf chlorosis while CLas inoculated US-942 and mock-inoculated plants displayed no growth reductions or foliar disease symptoms.

Sydney hybrid 72&74
Sydney hybrid (Microcitrus virgata) is a hybrid of Microcitrus australis 9 M. australasica (Reuther et al., 1967). Seedlings of Sydney hybrid with different HLB disease responses were provided by Ramadugu. Syd72 was HLB-tolerant and Syd74 was susceptible in controlled greenhouse assays where the seedlings were exposed to CLas containing psyllids in a no-choice situation for two weeks. Symptom expression and CLas titre were monitored for an extended period of time and the disease response phenotype was determined.

Nb material, CLso inoculation and VIGS
Nb plants were grown in a plant growth room with 12-h light/12h dark photoperiod at 23 AE 1°C. 10-day-old Nb plants (with 2 true leaves emerged) were exposed to 10 CLso-positive nymphs of potato psyllid for five days. After three days recovery, VIGS was performed by infiltration with A. tumefaciens strain GV3101 containing the bipartite TRV, pTRV1 and pTRV2 vectors (Liu et al., 2002). Equal volumes of A. tumefaciens (OD 600 = 0.5) suspensions containing pTRV1 and pTRV2-derived constructs or pTRV2-siRB non-target vector were mixed before infiltration (Song et al., 2003). The pTRV1-and pTRV2-carrying A. tumefaciens were then co-inoculated into the third and fourth true leaves of the plant by infiltration with 1ml syringe. The pTRV2-siBRAP and pTRV2-siVAD construct which can silence NbBRAP and NbBVAD, respectively, was designed by the SGN VIGS Tool (Fernandez-Pozo et al., 2015) and the primer sets used for amplifying the fragment are shown in Table S10. The leaves of inoculated plants were collected after three weeks for DNA and RNA extraction.

RNA preparation and qRT-PCR
Citrus Total RNA was extracted from ground tissue using acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski et al., 1987), followed by phenol/chloroform/isoamylalcohol extraction and precipitation with isopropanol.
Nb total RNA was extracted using TRIzol Reagent (Invitrogen) and treated with DNase I (Roche). Total RNA was reversetranscribed using SuperScript III reverse transcriptase (Invitrogen) with small RNA specific RT primers or oligo(dT) primer (Table S10). For qPCR, transcripts were amplified from 2 lL of a 20 9 diluted cDNA and iQ SYBR Green Supermix (Bio-Rad). The PCR amplification consisted of 3 min at 94°C, 45 cycles of 30 s at 94°C, 30 s at 62°C and 1 min at 72°C, 15 min at 72°C, followed by the generation of a dissociation curve. The generated threshold cycle (CT) was used to calculate the transcript abundance relative to Nb Ubiquitin (NbUbi) (Jin et al., 2002) or Citrus Actin (CsAct) (Zhao et al., 2013) (Table S10).

Small RNA library construction and sequence analysis
Total RNA was resolved by a denaturing 14% polyacrylamide gel, from which 18-28 nt RNA fragments were recovered. The purified fragments were ligated to a 5' adaptor (GUUCAGA-GUUCUACAGUCCGACGAUCAG), and a 3' adaptor (UCGUAUGCCGUCUUCUGCUUG) according to the Illumina small RNA-seq library prep kit protocol. The ligated fragments were gel-purified and reverse-transcribed by SuperScript III (Invitrogen, Carlsbad, CA, US). After PCR amplification for 15 cycles with Phusion High-Fidelity DNA Polymerase (ThermoFisher, Waltham, Ma, US), the PCR products were gel-purified and sequenced according to the Illumina sRNA sequencing protocol. Total mRNA or sRNA-enriched preparations were used to generate cDNA fragment libraries with the respective Illumina kits for the Illumina HiSeq-2000 platform. The adaptor-trimmed and quality-filtered sRNAs were clustered based on their mapping positions. The number of reads normalized to total reads was used to estimate their expression. Conserved miRNAs were identified by sequence similarity searches against the registry of plant microRNAs in miRBase (Kozomara and Griffiths-Jones, 2014). The targets of conserved and novel citrus miRNAs or siRNAs were then predicted by psRNATarget against the transcript library of Citrus clementine (JGI genomic project, Phytozome 11 182 v1.0; Dai et al., 2018). DAVID bioinformatics resources was used for the Gene Ontology (GO) enrichment analysis of the target genes (Huang et al., 2009a(Huang et al., , 2009b.

CLso detection by real-time PCR
To detect CLso in Nb plants, plant DNA was purified following the CTAB (cetyltrimethyl ammonium bromide) protocol from the midvein of leaves (Murray and Thompson, 1980). Then, CLso was detected by quantitative real-time PCR in the plant using the primers, TaqMan probe designed against CLso 16S rDNA (Table S10). The primers and probe of the positive internal control set were designed on basis of the ubiquitin gene from Nb (NbUbi). The amplification protocol of quantitative PCR was set at 40 cycles with 95°C for 10 min and 60°C for 30 min. Dilutions of plasmid DNA pLso (containing target DNA sequence from CLso in pGEM-T easy vector, Promega) were used to set a standard curve for evaluating the titre of CLas. L.N. and C.B. carried out the experiment. C.R. provide the citrus material. C.H. wrote the manuscript with support from H.J., J.T., U.A., C.R. and K.B. U.A., K. B.. All authors read and approved of its content.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Figure S1. The GO enrichment analysis of US-942 small RNA target genes reveals that regulators involved in defence responses may contribute to the HLB-tolerance of US-942. Figure S2. HLB-tolerant Syd72 has higher expression level of miR399 than HLB-susceptible Syd74. Figure S3. The expression pattern of four selected sRNAs (Figure 2) in infected Cleopatra and US-942 plants was measured at 7 and 28 weeks post inoculation (wpi). Figure S4. The expression pattern of the target genes of the selected sRNA regulators (Figure 2) was measured in infected Cleopatra and US-942 plants at 7 and 28 wpi. Figure S5. Disease development of Nb plants was shown at two weeks after infection with CLso by potato psyllid nymph transmission. Table S1. miRNAs that are differentially expressed in US-942 and Cleopatra. Table S2. Small RNAs that have higher constitutive expression levels in US-942 compared to Cleopatra. Table S3. Predicted target genes of sRNAs in Table S2. Table S4. Small RNAs that have lower constitutive expression levels in US-942 compared to Cleopatra. Table S5. Predicted target genes of Small RNAs in Table S4. Table S6. Small RNAs up-regulated after CLas infection in US-942 but not in Cleopatra. Table S7. Small RNAs down-regulated after CLas infection in US-942 but not in Cleopatra. Table S8. Predicted target genes of the top 50 small RNAs in Table S6. Table S9. Predicted target genes of the top 50 small RNAs in Table S7. Table S10. Primers used in this study.


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Feeding farm animals based on the new innovative total mixed ration (TMR) technology

The main part of the meat products produced in the country is beef. Currently, the number of cattle is over 12 million, of which 94% are livestock, and 90% are meat products. The efficient use of livestock feed plays an important role in improving digestion and increasing productivity. Feeding the animals with Korean total mixed ration (TMR) technology leads to improved feed efficiency and, at the same time, increased productivity. In this regard, research work on the application and implementation of TMR technology in farms of the population of Uzbekistan for cattle is relevant. This article presents the results of studies of feeding using Korean technology in households.

Introduction
Adequate feeding of farm animals is one of the main factors in increasing the productivity of animal husbandry. The meat productivity of farm animals varies depending on their breed, sex, age and genetic characteristics, and most importantly -on the feeding rate.
When assessing the quality of meat, the ratio of muscle and adipose tissue is assessed. The protein content of beef ranges from 15.75% to 24.8%, and the quality of the meat is determined by the ratio of the amino acid tryptophan to the amino acid hydroxyproline. The less hydroxyproline in meat, the better the quality of the meat. When the ratio of tryptophan and hydroxyproline is 4.5-5.0, the meat is considered to be of high quality [1][2][3]. The tenderness and pleasant taste of meat is determined by the amount of fat accumulated in the range of muscle tissue [4,5], the percentage of adipose tissue in meat can be up to 10-45%, depending on the age, sex and feeding rate of the animal. Another key metric is the meat to bone ratio. As the age and fatness of cattle increases, the percentage of bones in meat decreases and averages 4:1, the calorie content of 1 kg of meat depends on the protein content of the meat, especially the fat between the musclesm [6]. The average calorie content of 1 kg of meat is 1230 kcal, and the average calorie content is 2880 kcal. The bulk of the mixed feed produced in the country is wheat bran, the nutritional value of which is much lower than that of cotton, soybeans, sunflower meal and corn, wheat, barley grains, the bulk of which is indigestible fiber. In this regard, the development of the most optimal and affordable methods of production of beef in farms and households is one of the main challenges facing the industry. To this end, scientific work is being carried out on the application of Korean technology in cooperation with the KOPIA center for  the development of intensive feeding technology using cheap feeds enriched with protein, vitamins  and minerals, as well as chopped juicy and roughage feeds mixed with compound feed. In 2015-2017, the Research Institute of Livestock and Poultry, in cooperation with the Korean KOPIA Center in Uzbekistan, conducted a study on feeding and fattening young cattle using Korean TMR technology. According to research, gobies raised using Korean technology had a higher live weight, as well as a higher quality of meat. Therefore, taking into account the fact that the bulk of the livestock now falls on the population, in the period 2018-2020. work is underway to introduce this technology at home. According to Nasyrov (2011) [7] and Bulyshev (1990) [8], the growth of calves slows down with age. Consequently, the amount of feed consumed per 1 kg of added weight also increases. For example, calves at the age of 1-3 months consume 3-4 kg of feed for a gain of 1 kg, for 6-month-old calves, 3.5-4 kg of feed are consumed for each kilogram of weight gain, and at the age of 12-18 months, 8-10 kg of feed. According to Ashirov (1994) [9], Watter (2009) [10], and Kuhn et al (2013) [11], the shorter the period of adaptation of animals to the type of feeding during the year, the more stable the assimilation of nutrients. This is because the nutrients that enter the animal's stomach and the enzymes that are produced in the stomach to digest food will also be constant. Scientist, Nasyrov (2011) [7], who created an advanced school in the republic for the production of high-quality and cheap beef, came to the conclusion that when sowing alfalfa and catch crops in saline areas after cotton, salinization is prevented, and the amount of humus in the soil increases by 0.39%, the level of phosphorus and potassium decreases to 3.7, 14.5 and 46 mg/kg, respectively. When organizing a crop rotation system for cotton and wheat, with subsequent transplants of mung bean, beans, alfalfa, intermediate forage crops and corn, an increase in humus in the soil is observed. According to Mukhammadiev et al. (2014) [12], an experimental version of wheat straw silage fermented with Aspergillus terreus Uz CF-74 for 20 days contained 25.6% fiber, 33.6% fiber in the control, the protein content in the experiment was -8.8%, in the control -3.2%, the amount of pH in the experiment -4.5%, in the control -5.8%. The expected productivity can be achieved by satisfying the need for protein in animal feeding. To do this, you can use protein-rich foods such as soybean meal, sunflower meal, fish meal, meat and bone meal, cotton meal, feed yeast, and artificial proteins to replenish the protein the animal needs.

Research Methodology
The main goal of the study is the effective use of feed, strengthening the fodder base, increasing the meat productivity of local livestock, organizing work on the production of high-quality and exportoriented meat on a scientific basis, increasing the income of the population and creating a meat breed in the republic. In 2018, research work began in the Republic of Karakalpakstan, Khorezm, Navoi, Bukhara, Tashkent, Fergana regions. Also in 2019, research work began in Surkhandarya, Kashkadarya, Syrdarya, Jizzakh, Namangan, Samarkand and Andijan regions. 30 households in each oblast were given 3 heads of young animals, one grass chopper, tanks for mixing feed and corresponding concentrates, and feeding the animals according to Korean TMR technology was established. Within the framework of the project, 1,170 heads of young cattle were transferred to 390 households in 13 regions, which will be fed for 20 months. The rations were developed in accordance with Korean feeding standards, taking into account the age, body weight and sex of the young cattle. In addition, high quality corn and triticale seeds are delivered to each household for cultivation, and advice on cultivation methods is provided. The main objectives of the study are: -organization of the use of Korean TMR technology when feeding young cattle at home; -preparation of cattle for fattening using TMR technology; -increasing meat productivity and improving the quality of cattle meat using TMR technology in households; -development of quality standards for meat of cattle raised using TMR technology; -creation of the foundations for the creation of beef cattle breeds in Uzbekistan using local cattle breeds, which will serve for the production of quality meat; -Organization of the cultivation of triticale and corn at home; -to increase the incomes of the population due to cattle feeding using TMR technology; -organization of seminars and trainings in each region in cooperation with Korean scientists; -to identify the most advanced households in feeding Korean cattle and recommend them to travel to the Republic of Korea to improve their qualifications.

Results and Discussion
For the research, 3 heads of experimental animals were purchased for 30 households in all regions of the republic (Table 1) When choosing households, they turned to the district and chose the area with the best livestock. On the recommendation of the regional khokimiyat, livestock buildings were identified and studied in turn. Agreements were signed with each selected household in cooperation with the institute and the KOPIA center. According to the terms of the contract, the selected household must have conditions for breeding cattle, the homeowner must have the skills and interest in animal husbandry, have land for planting corn and triticale, provide experimental livestock with grass feed, share information with the information received, adhere to the specified technologies, etc. In households, the main dimensions of young cattle were taken into account (height at the withers, oblique body length, chest circumference). After 6 months of experiments, the highest live weight in animals was observed in the Fergana region. Their average live weight was 188.2 kg, and the average live weight for the project was 177.9 kg. When analyzing the increase in live weight by region for 6 months. As a result of the analysis of the increase in live weight of animals during 6 months in animal households of the Republic of Karakalpakstan, an average of 74.7 kg, and the daily gain of 416 g, while the average daily gain of calves in the farms of the Khorezm region was 83.5 kg, and the daily gain -463 g, in the Navoi region the average daily gain of calves was 87.7 kg, the daily gain of 486 g, the average daily gain of calves in the farms of the Fergana region is 96.4 kg, the daily gain of 533 g, in the farms of the Tashkent region this figure was respectively 97.3 kg. and 540 BC The following types of feed were used for feeding cattle (Table 2). When studying the meat productivity of cattle raised using Korean technology for 20 months, a bull with an average live weight was selected, whose meat yield was 421 kg, slaughter yield was 55%. A comparative study of the cross-section of the 11th and 12th ribs using the Korean technology and the cross-section of the 5th and 6th ribs using the Japanese technology revealed the formation of fat between the muscle fibers, that is, the formation of marble pulp. The study also analyzed economic indicators (Table 3). Thus, the  A cost-benefit analysis of local and Korean feeding techniques is shown in the diagram below.

Conclusions
So, according to the research results, it was found that feeding using the Korean technology is effective. When feeding cattle using the new Korean technology, the profitability was 45.9%, and the resource saving factor was 1.05. Currently, the institute is working on breeding breeding bulls for selection work, selected as a result of selection from local cattle. These bulls will be used to create meat breeds of animals in the future and provide quality meat to the population.


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SUSCEPTIBILITY OF DIFFERENT STORED PULSES INFESTED BY PULSE BEETLE, CALLOSOBRUCHUS CHINENSIS (LIN.)

The susceptibility of different stored pulses infested by Callosobruchus chinensis L. (Coleoptera: Bruchidae) was observed in three varieties of stored pulses i.e. gram (Cicer arietnum L.), pea (Pisum sativum L.) and mung (Vigna radiata L.) during April, 2017 to May, 2018. The longest incubation-, larvaland pupal period of pulse beetle were in gram 5.4 ± 0.29, 12.6 ± 0.25, 5.5 ± 0.32 days, respectively and the shortest were in mung 4.6 ± 0.25, 11.3 ± 0.28, 4.2 ± 0.19 days, respectively. The shortest developmental period of pulse beetle was 20.1 ± 0.46 days in mung. The incubation-, larval-, pupaland total developmental period of pulse beetle varied significantly between the pulse beetle grown in gram and mung (p < 0.05). The highest longevity and fecundity were in gram, 8.2 ± 0.33 days and 70.2 ± 7.53, respectively. The fecundity did not vary significantly in different stored pulses (p > 0.05). The longevity and number of adults emerged varied significantly (p < 0.05) between the gram and pea. The number of male emerged did not vary significantly (p > 0.05) between the different stored pulses and the number of female emerged varied significantly (p < 0.05) between gram and pea, pea and mung. The experiment revealed that pulse beetle preferred smooth coated and large size seeds to oviposit. Pea was found to be most resistant to attack by C. chinensis L. whereas mung was more susceptible than gram and pea.

Introduction
Tremendous losses of the crops (pre-and post-harvest commodities) always occurred during storage due to bruchids. Among the various pest of stored pulses Callosobruchus (syn: Bruchus) are renowned as the major pest and serious damaging insects (1,2) . Varma et al. (3) have estimated that 15% damage of gram occurred by Bruchus chinensis and B. theobroma. The initial infestation started in the field itself, where adult female laid shiny bright yellow eggs, which were singly glued to seed surface and hatched within 3-5 days. The larvae were creamy colored, "C" shaped that bore into the pulse grain. Pupa were obtect type and also creamy colored. The larval and pupal period varied from 13 -20 days. The entire development took place inside the developing seed and adult emerged *Author for correspondence: <shefali@du.ac.bd>. out leaving behind holed grains (3)(4)(5) . Their oviposition and growth rate were continuous and need 3 -4 months for total destruction of the seeds and in the meantime the pest rapidly increased their population (6) . Pulse have a prominent place in daily diet and contains 20 -30% proteins, supply a good production of carbohydrates, minerals and fats (7) . Protection of pulses from these pests is one of the major problem. In developing countries, 12-30% damages are caused due to Callosobruchus sp. because they feed on the protein content of the grains (8) . Proliferation of insect pests occur during storage due to high moisture content of grains (>12%), high temperature (25 -35℃) and relative humidity (>60%) (9) . Less than 10% moisture content in pulse grain can reduce the infestation rate (10) .
In tropics, traditional farmers stored large quantity of food grains under one roof and that may lead to cross infestation among the stored products, which share common pests (11) . According to Gugar and Yadav (12) , 55 -60% weight and 45.50 -66.30% protein content losses occurred due to damage caused by pulse beetle. A single larva can destroy several mature seeds and severe infestation leads to 100% damage (13) . These losses often become enormous and cannot be neglected. Nineteen insect species have been recorded so far from stored grains in Bangladesh (14) . Low-yielding indigenous pulses are cultivated in different parts of the country that are highly susceptible to disease and insect pest. Agricultural stored products are infested 10 -40% annually by insect pest that causes economic loss in Bangladesh (15) .
From 2015 -2016, the production of pulse is nearly 378 metric tons per thousand acres in Bangladesh (16) . This huge amount of pulses need protection from insect pests. Heavy infestation on grains found to cause weight loss, decrease germination capacity, reduce commercial value of seeds and grains become unfit for human consumption (17) . Pulse beetle is an internal feeder of grains. Treatment of pesticide with food grain is not advisable, because it leads to several health hazards to human being, wildlife and environment. To prevent a heavy build-up of Callososbruchus sp. population it is necessary to gain the knowledge about host, pest and environment interaction. The present study was undertaken to determine the susceptibility and suitability as well as to investigate the biology and biotic performance of C. chinensis L. that can help to find out a easy way to control method of this pest.

Materials and Methods
The experiments were conducted by using three types of stored pulses viz., gram (Cicer arietnum L.), pea (Pisum sativum L.) and mung (Vigna radiata L.) and the life cycle and biotic performances of C. chinensis L. on these pulses were observed. The experiments were conducted in the Entomology Laboratory, Department of Zoology, University of Dhaka during April, 2017 to May, 2018. Experiments were carried out within an incubator at 30°C and 75% relative humidity (RH).
Pulse beetles (C. chinensis L.) were collected from Polashi bazar, Dhaka where the various types of pulses were stored. Pulse beetles were identified following the method used by Southgate et al. (18) , Southgate (19) and Rahman (20) . The cracked or damaged pulse grains were removed from each of the three pulse grain varieties and only fresh pulse grains were used as test samples. These pulses were washed thoroughly by tap water and then dried under sunlight. These pulses were deep freezed for about 24 hours to eliminate possible insect contaminations. Gram was selected as rearing means for stock culture and beetles were reared for three generations before they were used in the experiments.
For observing the life cycle of C. chinensis L. one hundred grains of each variety were taken in the Petri dishes and 5 pairs of male and female C. chinensis L. were released into each Petri dish. Then Petri dishes were kept into incubator at 30°C and 75% relative humidity for 24 hours. After 24 hrs the beetles were removed from the Petri dishes and the Petri dishes were again kept into the incubator at same temperature and humidity. The samples were observed daily. Experiments were replicated ten times for each variety that was marked as Rep. Experiment for biotic performances was replicated five times for each type of pulses. Fifty pulse grains from each type of pulse (gram, pea, mung) were kept in separate Petri dish. One pair of newly emerged male and female adults were released in each Petri dish. The Petri dishes were kept into incubator at 30°C and 75% RH. Released beetles were allowed to oviposit for two days. After two days, each pairs of beetles from each replication were transferred to fresh grains and were allowed to lay eggs again. The same transferring processes of male and female beetles were carried out after every two days till the death of adult. After the test all pulses were collected and the pulses containing eggs were separated out by examining under magnifying glass and the number of eggs laid by each female during her life time was recorded to study the fecundity. The pulses (gram, pea, mung) on which the beetles laid eggs were transferred into another sets of Petri dishes and observed daily until the adults emerged. After emergence the number of male and female adults were recorded.
Data obtained from the experiment were analyzed by one-way ANOVA. To evaluate the relative effectiveness of life cycle and biotic performance of pulse beetles on different type of pulses, the experiment data were subjected to t-test for significance in their differences. In all cases, a significance level of p < 0.05 was used.

Results and Discussion
The life cycle of Callosobruchus chinensis L. including incubation-, larval-, pupal-and total developmental period of all the three different stored pulses are shown in Table 1.
Adult females laid eggs on surface of the grains and they were attached singly with sticky substance. Eggs were cigar shaped and yellow in color. The longest incubation period was observed in gram, 5.4 ± 0.29 days and the shortest period being observed in mung, 4.6 ± 0.25 days. The incubation period of pulse beetle varied significantly between gram and pea (t = 0.04911), gram and mung (t = 0.03204) but did not vary significantly between pea and mung (t = 0.39574) (p > 0.05) ( Table 1). Almost similar observation of incubation period was reported by Patel et al. (1) in gram, pea and mung bean as 5.03, 5.53 and 4.53 days. The larval and pupal periods passed inside the seed. The larvae were yellow-whitish and pupae were dark brown in color. The longest larval and pupal period were observed in gram, 12.6 ± 0.25 and 5.5 ± 0.32 days, respectively and the shortest larval in mung, 11.3 ± 0.28 and 4.2 ± 0.19 days, respectively. The penetration ability of larva to the seed coat is influenced by the physical properties of the seed coat such as thickness, hardness, roughness (21) . The larval period varied significantly between gram and pea (t = 0.03129), gram and mung (t = 0.00227) and the pupal period were also varied significantly between gram and pea (t = 0.01029), gram and mung (t = 0.00206) (p < 0.05). But the larval and pupal period did not vary significantly between pea and mung (p > 0.05) ( Table 1).
The total developmental period of C. chinensis was longest in gram 23.5 ± 0.59 days and shortest in mung 20.1 ± 0.46 days that are comparable to Patel et al. (1) who recorded the total developmental period 23.49 days on pea and 17.19 days on mung. Among these three pulses, the rate of development was slower in gram. The total developmental period varied significantly between gram and pea (t = 0.00622), gram and mung (t = 0.00014) (p < 0.05) but between pea and mung (t = 0.22595) it did not vary significantly (p > 0.05) ( Table 1). According to Varma et al. (3) total developmental period varied from 22 -28 days.
The results of the present study corporate with the results obtained by Singh et al. (22) who reported the incubation period of C. chinensis L. was 4 -5 days and total developmental period 17.41 days. However, Howe et al. (13) observed the incubation and total development period and those were 4.2 and 18 -22.5 days, respectively. Raina (23) reported that the incubation period and larval and pupal period were 3.5 and 18 days, respectively. Among different pulses, the developmental responses varied. Differences between the duration of developmental stages may vary due to temperature, humidity and different types of pulses (1,24,25) .
The biotic performances of C. chinensis L. in all the three different stored pulses are shown in Table 2. The fecundity was highest in gram 70.2 ± 7.53 and lowest in pea 55.4 ± 3.73. The fecundity did not vary significantly between gram and pea (t = 0.07695), pea and mung (t = 0.17174), gram and mung (t = 0.40731) (p > 0.05). The number of adult emerged was highest in gram 65 ± 7.58 and lowest in pea 41.8 ± 1.93 and the number of male and female emerged was 33 ± 4.47 male and 32.2 ± 3.42 female in gram, 23.6 ± 1.19 male and 18.2 ± 1.25 female in pea, 27.6 ± 6.50 male and 23.8 ± 2.30 in mung. Adult emergence was highest in gram, closely followed by mung bean. The number of adults emerged varied significantly between gram and pea (t = 0.01401) (p < 0.05), but did not vary significantly between gram and mung (t = 0.15457), pea and mung (t = 0.17603) (p > 0.05). The number of male emerged did not vary significantly between gram and pea (t = 0.0533), gram and mung (t = 0.27864), pea and mung (t = 0.30138) (p > 0.05). The number of female emerged varied significantly between gram and pea (t = 0.00435), pea and mung (t = 0.04613), but did not vary significantly between gram and mung (t = 0.052703) (p > 0.05). The longevity of the adult was longest in gram 8.2 ± 0.33 days and closely followed by mung 7.8 ± 0.33 days and shortest in pea 7.0 ± 0.28 days. The longevity of the adults varied significantly between gram and pea (t = 0.01998) (p < 0.05), but did not vary significantly between gram and mung (t = 0.2356), pea and mung (t = 0.07055) (p > 0.05) ( Table 2). Raina (23) reported that the longevity of male and female were 9.76 and 9.44 days that are comparable with the present findings. In the present study, it was observed that the adults of C. chinensis preferred to oviposit on smooth coated and large sized seeds. Wijenayake et al. (26) reported that the beetles preferred smooth coated seeds for oviposition and rejected seeds with rough coat. But smoothness of the seed coat is not the only factor for higher oviposition. There is a combination of factor such as seed texture, weight and volume of seed, size and shape, seed color that are responsible for ovipositional performance of bruchids (21,27) .
Considerable numbers of eggs were oviposit on all seeds. According to Yadav and Pant (28) Callosobruchus spp. will oviposit on any seed, even though the seed may not be suitable for the development of the young. Grish (29) reported that C. maculatus oviposition performances had been shown towards the smoothness of seed coat and size of the grain.
From the present study, it can be concluded that mung was more susceptible than others to complete the life cycle of C. chinensis. Biotic performance of pulse beetle on pea was very poor, hence it can be considered relatively resistant to attack by pulse beetle. Among the three pulses, mung was more susceptible and pea found to be more resistant to attack by C. chinensis. Farmers should not store mung with other pulses in the same place to avoid cross infestation for their high susceptibility to C. chinensis. To prevent the infestation of pulses or to take any prerequisite it is necessary to know the susceptibility of different pulses.


extfieldsofstudy:
Physics
Environmental Science

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Experimental study on gas production optimization of pig manure and wheat straw under different pig manure levels

In order to explore the optimal ratio of pig manure and wheat straw mixed fermentation process, the experiment controlled the temperature of the fermentation control fermentation process to 35 °C, the total mass of the fermentation liquid was 700 g, and the total soild (TS) was 8%. The effect of pig manure content on fermentation. Based on the analysis of the gas production per unit of TS, when the pig manure content is 80%, the unit TS produced has the highest gas yield of 344 ml/g, which is 58.9% higher than that of pure wheat straw fermentation. Fermentation increased by 11.43%. In order to explore the best pig manure content in the fermentation process, regression analysis was performed on the gas production per unit of TS by origin. The result was that when the content of pig manure was 79%, the gas production was the highest, 336.3 ml/g, which was higher than that of wheat straw fermentation. 55.51%, 9.05% higher than pure pig manure fermentation. The methane content was used as the analysis result: when the pig manure content was 75%, the highest average methane content after start-up was 60.33%, which was 15.24% higher than that of wheat straw alone, and 3.4% higher than that of pig manure alone. Therefore, during the fermentation process of pig manure and wheat straw, when the content of pig manure is controlled at about 75%, the biogas production in the fermentation process and the methane content in the biogas can be effectively improved..

Introduction
According to the China Statistical Yearbook 2018, China's pig breeding has a net output of 702 million heads, and the annual planting area of wheat is 24,500,000 hectares [1] . At present, livestock manure is mainly treated as organic fertilizer and returned to the field after simple treatment. The method of straw utilization mainly includes making fuel and crushing the field [2,3] . When livestock manure is used as organic fertilizer, the stench or sewage generated is likely to have a serious impact on the living of the surrounding residents; when the shaped fuel is produced, the energy consumption is high, and the smoke after combustion also has an impact on the atmospheric gas production. The process of returning to the field is due to the slow decomposition of microorganisms in the ground, and the problem of pests after returning to the field is becoming more and more serious. Biogas fermentation uses organic matter such as livestock and poultry manure, crop straw, and kitchen waste as fermentation raw materials. The raw materials are abundant, and the energy consumption of the fermentation process is low. The biogas is clean renewable energy, and the biogas residue is highquality organic fertilizer. It is an effective way to integrate energy and environmental protection [4,5] . However, the current efficiency of biogas fermentation is still not high, which restricts the large-scale promotion of biogas [6] . Straw in crop straw is composed of lignocellulose and hemicellulose. The C/N of wheat is generally high [7,8] , and the C/N of livestock manure is generally low, which is also not suitable for microorganisms in fermentation process requirements for C/N. Therefore, many domestic and foreign scholars have done a lot of research on biogas mixed fermentation.
Weiland [9] mixed fermentation of different raw materials, and the fermentation efficiency of the mixed raw materials was higher than that of the single raw materials. Jiang et al [10] studied the best gas production when cow dung and corn stalks were 2:1. Parkin et al [11] found that when the C/N ratio is between 20 and 30, the gas production effect is better, and the C/N control is at 25, and the gas production effect is the best. Lehtomä ki et al [12] used different straw and cow dung for mixed anaerobic fermentation. When the content of straw accounted for 30%, the yield per unit volume of methane increased by 16%-65% compared with the fermentation of cow manure alone. Wang et al [13] in the continuous fermentation reactor at 55 ° C, adding 1t pig manure and 4.6kg wheat straw for fermentation, the results showed that the methane content in the mixed fermentation was 10% higher than the methane content in the pig manure fermentation alone. Chen et al [14] found in the study of pig manure and rice straw, adding pig manure to the straw can greatly increase the ammonia nitrogen content in the fermentation feed liquid, which is conducive to the smooth progress of fermentation. Liu et al [15] studied the anaerobic fermentation effect of the ratio of manure to grass in dry fermentation at 35 °C. The peak gas production of fermented pig manure and rice straw mixed fermentation appeared 11 to 15 days earlier than the separate fermentation of manure and stalk. The peak gas production is also increased by 85~265ml.
The above experiments only analyze the gas produced in the mixed fermentation, and the research on the methane content in the mixed fermentation is less. In this experiment, pig manure and wheat straw were used as fermentation raw materials. Based on the unit gas production rate and methane content, polynomial fitting was used to explore the optimal ratio of pig manure and wheat straw in mixed fermentation, and provide data reference for biogas fermentation engineering operation.

Experimental materials and pretreatment
In the experiment, pig manure and wheat straw were used as fermentation raw materials, and the normal fermentation of the biogas slurry in the laboratory was used as an inoculum. The pig manure was taken from the Zhengzhou breeding farm in Henan Province. After collection, it was naturally airdried and stored for use in the experimental process. The straw was from the third living experiment area of Henan Agricultural University. After the straw was collected, it was crushed to a length of 5-10 mm and added to the biogas slurry. The clear liquid is made to have a water content of about 70%, and is stacked, and the stacking process is stirred once a day until the surface of the straw grows white hyphae. The C/N, TS and VS contents of each raw material and inoculum before the experiment are as shown in Table 1.

Experimental device
The experimental device is composed of a thermostat device, a fermentation device and a gas collecting device. The thermostat is provided by a 250 L incubator in the laboratory. The fermentation device consists of a 1000 ml volumetric flask, the bottle mouth is sealed with a rubber stopper, a rubber stopper and a conduit; the gas collection device is collected by a drainage gas collection method. A conduit link is used between the fermentation unit and the gas collection unit, as shown in

Experimental design
The experiment used pig manure and wheat straw as fermentation raw materials. The experiment was divided into 8 groups, and each group was set up in two parallels. In the 8 groups of experiments, pig manure and wheat straw were used as raw materials for separate fermentation and pig manure and wheat straw were mixed and fermented according to pig manure content. The materials of each group were shown in Table 2. The test adopts a feeding method to add pig manure and wheat straw to the fermentation bottle according to a certain dry matter ratio, and 210 g of the inoculum is added to the fermenter, and the total mass of the fermentation liquid is 700 g with distilled water, and the total TS content is 8%. The pH value, gas production and corresponding methane content of the fermented feed liquid were measured regularly every day, and the average value was used as the result for analysis.

Test items and methods
The total solids and volatile solids were determined by drying and burning. The nitrogen (N) and carbon (C) contents were determined using a C-N elemental analysis (vario EL cube). pH was measured using a pH meter (PHS-3E). Gas yields were measured using drainage gas gathering method. Methane content was measured by gas chromatography (Agilent 6820) equipped with a FID detector  4 and a carbosieve S II packed column. Nitrogen as carrier gas and the carrier gas, The temperature of carrier gas, column and the detector were adopted 60℃,80℃ and 150℃ respectively.

Changes in methane content of each group during fermentation
The variation trend of methane content in each group during fermentation was as shown in Figure.2. During the first five days of fermentation, the methane content of each group increased rapidly, reaching about 40%, and then the rate of increase of methane content slowed down, about 15 days after fermentation. During the fermentation process, the peak gas production of each group and the average methane content of the whole process and the methane content after startup are shown in Table 3. When the pig manure content is lower than 66%, the peak value of methane in each group increases with the increase of pig manure content. When the pig manure content is above 66%, the peak value of methane in each group is basically stable at about 70%. The peak value of methane in pure straw fermentation was 58.2%, and the peak value of methane in pure pig manure fermentation was 74.08%. The average methane content after start-up (A methane content of 50% is considered to be activated), when the pig manure content is less than 50%, the average methane content increases with the increase of pig manure. When the pig manure content is above 50%, the methane content of each group after start-up is around 59%. Which is the highest when the pig manure content is 75%, which is 60.33%, 15.24% higher than the pure wheat straw fermentation (52.35%), and 3.4% higher than the pig manure (58.34%). It shows that the mixed fermentation of pig manure and wheat straw in a certain ratio can increase the methane content of fermentation.
Table3. Peak methane content, average methane content, and average methane content after start-up. Groups

Changes in gas production of each group during fermentation
3.2.1. Trends in daily production rate of each group. The gas production rate is the most direct manifestation of fermentation. The higher the gas production rate, the shorter the fermentation cycle and the higher the fermentation efficiency. Figure 3 shows the trend of daily gas production rate during the fermentation process. During the whole fermentation process, there are two distinct gas production peaks in the daily gas production rate of each group. But there are some differences in the peak of gas production in different groups. The peak of gas production in each group appeared for the first time on the 3rd to 7th day of fermentation, and the second time on the 12th to 18th day of fermentation. The R1 group is pure straw fermentation, the maximum gas production rate is 800ml/d, the pig manure content in the R2 and R3 groups is less than 50%, and the peak value is relatively low, 825 ml/d and 1050 ml/d, respectively, and the peak appears relatively late. The pig manure content in the R4, R5, R6 and R7 groups is between 50% and 80%, and the maximum gas production rate is relatively high, respectively 1580 ml/d, 1350 ml/d, 1500 ml/ d, 1395ml / d, the peak appeared relatively early, on the sixth and seventh days of fermentation, R8 is pure pig manure fermentation, the maximum gas production rate is 1470ml / d. The peak gas production and time of each group are shown in Table 4. Table 4 shows that when the pig manure content is less than 50%, the gas production peak is lower, and the peak appears later. When the pig manure content is above 50%, the gas production peak is relatively high, and the occurrence time is earlier. It is indicated that an appropriate increase in pig manure content can increase the peak gas production and the peak time.  Figure 3. Trend of daily gas production rate during fermentation in each group. Table 4 Gas production peak and corresponding time.

Groups
First peak (ml) First peak time (d) Second peak (ml) Second time (d)  R1  740  5  800  12  R2  415  3  825  13  R3  900  7  1050  14  R4  1585  6  1125  13  R5  1350  6  1280  15  R6  1500  7  1400  15  R7  1395  6  890  18  R8  1470  6  1150  15 3.2.2. Trends in cumulative gas production and trends in unit TS. Figure 4 and Figure 5 shows the trend of cumulative gas production during fermentation and the gas production per unit TS of each group. In the first five days of fermentation, the cumulative gas production of each group began to rise rapidly. After, the cumulative gas production trend between different groups changed significantly. The cumulative gas production of R1, R2 and R3 is relatively slow, and the rate of R5, R6 and R7 rising is relatively fast. After 18 days, the gas production rate decreased, and the cumulative gas production rate of each group gradually slowed down until the end of fermentation. During the whole fermentation process, the final cumulative gas production of R5, R6 and R7 is relatively high, and R1 is the lowest.
The total gas production of pure wheat straw (R1) fermentation process is 12110 ml, the gas production per unit TS is 216ml/g; the total gas production of pure pig manure (R8) fermentation is 17270 ml, and the gas production per unit TS is 308ml/g. During the mixed fermentation of pig manure and wheat straw, the total gas production of each group increased with the increase of pig manure content. When the pig manure content was 80%, the total gas production was the highest, being 19245 ml, and the corresponding unit TS gas production was 344ml. /g, the total gas production by pure wheat straw fermentation increased by 58.9%, which was 11.43% higher than the total gas production of pig manure alone. It indicates that increasing the content of pig manure during mixed fermentation can increase the cumulative gas production of mixed fermentation of pig manure and wheat straw.   Figure 4. Trend of cumulative gas production Figure 5. Unit TS gas production of each groups. in each group during fermentation.

Polynomial fitting of unit TS gas production.
In order to obtain the optimal ratio of the fermentation process of pig manure and wheat straw more accurately, the final unit gas production of the fermentation was analyzed by regression analysis. Figure 7 and Figure 8 show the quadratic polynomial fitting and cubic polynomial fitting of the unit TS gas production. The closer R 2 is to 1 during the polynomial fitting process, the higher the degree of fit.
The fitting results are shown in Table 5, where X is the pig manure content and Y is the total gas production. Table 5 shows that the R 2 of the quadratic polynomial fitting model is 0.91. When the content of pig manure is 87%, the highest unit TS gas production rate is 326.69ml/g; the R 2 of the cubic polynomial fitting model is 0.9938, the whole model had the largest unit TS gas production rate of 336.3 ml/g when the pig manure content was 79%. Compared with the quadratic fitting, the R 2 of the cubic term fit is closer to 1, resulting in higher reliability.  Figure 6. Quadratic fit curve. Figure 7. Cubic fitting curve.

Conclusions
(1)Mixed raw material fermentation can improve gas production efficiency. Mixed fermentation process, the unit TS gas production rate of each group increased as the pig manure content in the fermentation material increased. When the pig manure content is 25%, the unit TS gas production rate is 250.94ml/g, which is 16.04% higher than the wheat straw fermentation alone, 18.62% lower than the pig manure fermentation alone; the mixed fermentation efficiency is the highest when the pig manure content is 80%. , it is 343.66ml/ g, which is 58.92% higher than the straw alone, and 11.44% higher than the pig manure alone. When the pig manure content is less than 50%, the peak appears first, but the peak value is also low. When the pig manure content is higher than 50%, the peak height does not differ significantly from the time of occurrence. Therefore, an appropriate increase in pig manure content can effectively increase the amount of gas produced by fermentation and the peak time of gas production.
(2)Fermentation of mixed raw materials can increase the methane content in the gas. When the pig manure content is less than 50%, the increase of pig manure content during the mixed fermentation process can increase the methane content in the fermentation process. When the pig manure content is higher than 50%, the methane content is relatively high, there is no significant difference. When the pig manure content is 75% after start-up, the average methane content is highest, which is 60.33%, and which is15.24% higher than that of wheat straw is alone, and 3.4% higher than that of pig manure alone.
(3)The best ratio of mixed raw materials. Binomial and trinomial fittings were performed on the unit TS gas production rate of each group using the original 8.0 software. The R 2 of the three terms is 0.9193, and the R 2 of the three-phase is 0.9938. The trinomial fit is better than the binomial fit. As a result of the trinomial fitting, when the content of pig manure was 79%, the fermentation had the best gas production rate of 336.3 ml/g, which were 55.51% higher than that of wheat straw fermentation and 9.05% higher than that of pig manure fermentation. Pig manure content (%)


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Influence Of Soybeans Seedling Thickness And Fertilization Norms On Cotton Yield

This article presents the results of a research of the influence of soybeans on the thickness of the seedling and fertilization norms on the cotton harvest

INTRODUCTION
It is known that one of the factors increasing the productivity of agricultural crops is the use of science-based crop rotation systems. The choice of past crops is important in crop rotation. Achieving food security, soil fertility and natural crop yields has also been proven in many years of experience to achieve direct crop rotation through the cultivation of legumes as a primary and secondary crop.

THE MAIN FINDINGS AND RESULTS
The influence of legumes on the change in the general and mobile forms of humus, nitrogen, phosphorus and potassium content in the soil from repeated crops planted after winter wheat has a special place. This is due to the fact that the amount of root residues in legumes, which quickly turn into various forms of nutrients, has a positive effect on soil fertility and increasing the yield of crops planted next year (A. ; opportunities to reduce annual nitrogen by 25-30% and potassium by 45-50% in the coming season; The tasks of increasing the yield of winter wheat by 15-17% and the yield of cotton by 10-12% in the cotton-grain rotation system have been studied [9, p. 66; 10].
The study of the effect of shade seedling thickness and fertilization standards on soil fertility and the growth, development and yield of cotton planted after them in the conditions of loamy soils further complements the above tasks.

RESEARCH METHODS
Experiments were conducted to study the effect of crop rotation on soil fertility and cotton yield in the shade system of cotton in the desert region of Kashkadarya region. The experiment was placed in 11 variants, 3 returns. The experiment was planted at 90 cm row spacing, with each option being 30 m long and located at 108 m2. The calculated area for phenological observations of accounting works is 54 m2, the total area is 0.39 ha.

RESULTS OF THE STUDY
It is known that the effectiveness of each applied agro-technical measure is determined by productivity. In our study, the yield was determined by weighing the weight of one bale of cotton, along with determining the yield before each harvest. The results obtained are shown in Figure 1.
According to the data obtained, along with the agronomic measures carried out in the cultivation of cotton in the past, the past crop, their seedling thickness and the weight of cotton in one pod under the influence of the norms of mineral fertilizers applied caused them to vary. In the 1st variant of the experiment, when control of cotton instead of cotton, the average weight of 1 piece of cotton in 2 years was 2.63 grams, in the 2nd variant the weight of 1 piece of cotton was 2.73 grams, the best indicator was 370 thousand bushes / ha of seedlings. fertilizer N100P120K100 kg / ha was observed in the normally applied variant and amounted to 2.91 grams, respectively. This was found to be 0.28 grams higher compared to the control option. From the variants of mineral fertilizers N100P120K100 kg / ha, it was found that the weight of cotton in the pods was 0.11-0.14 grams higher than the control in the cultivars with a seedling thickness of 222.0 and 270 The American Journal of Agriculture and Boimedical Engineering (ISSN -2689-1018) Published: December 29, 2020 | Pages: 28-33 Doi: https://doi.org/10.37547/tajabe/Volume02Issue12-06 IMPACT FACTOR 2020: 5. 34 OCLC -1121105746 thousand bushes per hectare. Soybean N75P100K75 kg / ha was found to be 0.09 g higher in cotton than the control in the cottonfed variant against the background of seedling thickness of 370,000 bushes / ha in the variants with mineral fertilizers in the norm, while in the remaining variants this figure was reduced.

Figure 1: The weight of cotton in a cocoon observed in the experiment, g
Harvest was carried out twice a season in the experimental field. Based on these data, the yield of cotton was determined by variants. According to him, the average yield of cotton in the control variant (option 1) was 35.3 ts / ha. The best indicator of the total cotton yield of cotton was observed in the variant of soybean application of mineral fertilizers N100P120K100 kg / ha, which left 370 thousand bushes / ha, respectively, and amounted to 45.7 ts / ha, respectively. In the remaining variants of the experiment, it was also noted that the yield was higher than the control. As an example, in 3-4 variants, the average in 2 harvests was 39.4 and 41.7 ts / ha, respectively.
Even in the variants of soybean N75P100K75 kg / ha with the application of mineral fertilizers, the yield of cotton was observed against the background of seedling thickness of 370 thousand bushes / ha in the cultivated variant, and the yield was 7.1 ts / ha higher than the control. These options reported lower results compared to the options that used high-dose fertilizer.
The lowest rate was observed in cotton growing after the cultivated variant, leaving 550 thousand bushes per hectare of soybeans in both backgrounds, the yield was 40.0 ts / ha against the background of high fertilizers and 36.0 ts / ha against the background of low fertilizers. The data are presented in Table 2.

CONCLUSION
From the results obtained, it can be concluded that the application of soybean mineral fertilizers at the rate of N100P120K100 kg / ha, leaving 370 thousand bushes per hectare of seedling thickness, and subsequent cultivation of cotton provides a basis for high yields of cotton.