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Effects of Plant Growth-Promoting Bacteria (PGPB) Inoculation on the Growth, Antioxidant Activity, Cu Uptake, and Bacterial Community Structure of Rape (Brassica napus L.) Grown in Cu-Contaminated Agricultural Soil

Previous analyses of plant growth-promoting bacteria (PGPB) combined with the remediation of heavy metal pollution in soil have largely been performed under potting or greenhouse conditions, and in situ remediation experiments under field conditions have rarely been reported. In this study, the effects of the metal-resistant PGPB Microbacterium oxydans JYC17, Pseudomonas thivervalensis Y1-3-9, and Burkholderia cepacia J62 on soil Cu pollution under rape remediation were studied in the farmland surrounding the Nanjing Jiuhuashan copper mining region in China. Following inoculation treatment for 50 days, the biomasses of the rape inoculated with strains JYC17, Y1-3-9, and J62 increased, and the total amounts of Cu uptake increased by 113.38, 66.26, and 67.91%, respectively, the translocation factor (TF) of rape inoculated with J62 was 0.85, a significant increase of 70.68%, thus improving the Cu remediation efficiency of the rape. Y1-3-9 and J62 affected the bioavailability of Cu in the soil, and the water-soluble Cu contents were increased by 10.13 and 41.77%, respectively, compared with the control. The antioxidant activities in the rape leaves showed that the tested bacteria increased the contents of antioxidant non-enzymatic substances, including ascorbic acid (ASA) and glutathione (GSH), which were increased by 40.24–91.22% and 9.89–17.67%, respectively, thereby reducing the oxidative stress caused by heavy metals and the contents of thiobarbituric acid-reactive substances (TBARS) and peroxidase (POD). PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the effects of the tested bacteria on the cultivation-dependent and cultivation-independent bacterial communities in the root endosphere and rhizosphere soil of the rape. The sequencing results of the DGGE bands indicated that the tested bacteria colonized the endosphere and rhizosphere, and they became an important component of the cultivation-dependent bacteria. The canonical correspondence analysis (CCA) of the DGGE profile and similarity cluster analysis showed that the tested bacteria affected the cultivation-dependent and cultivation-independent bacterial communities in the root endosphere and rhizosphere. In this experiment, the effects and mechanisms of the combined plant-microbe remediation under field conditions were preliminarily studied, and the results are expected to provide a theoretical basis for future combined remediation experiments.

Previous analyses of plant growth-promoting bacteria (PGPB) combined with the remediation of heavy metal pollution in soil have largely been performed under potting or greenhouse conditions, and in situ remediation experiments under field conditions have rarely been reported. In this study, the effects of the metal-resistant PGPB Microbacterium oxydans JYC17, Pseudomonas thivervalensis Y1-3-9, and Burkholderia cepacia J62 on soil Cu pollution under rape remediation were studied in the farmland surrounding the Nanjing Jiuhuashan copper mining region in China. Following inoculation treatment for 50 days, the biomasses of the rape inoculated with strains JYC17, Y1-3-9, and J62 increased, and the total amounts of Cu uptake increased by 113.38, 66.26, and 67.91%, respectively, the translocation factor (TF) of rape inoculated with J62 was 0.85, a significant increase of 70.68%, thus improving the Cu remediation efficiency of the rape. Y1-3-9 and J62 affected the bioavailability of Cu in the soil, and the water-soluble Cu contents were increased by 10.13 and 41.77%, respectively, compared with the control. The antioxidant activities in the rape leaves showed that the tested bacteria increased the contents of antioxidant non-enzymatic substances, including ascorbic acid (ASA) and glutathione (GSH), which were increased by 40.24-91.22% and 9.89-17.67%, respectively, thereby reducing the oxidative stress caused by heavy metals and the contents of thiobarbituric acid-reactive substances (TBARS) and peroxidase (POD). PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the effects of the tested bacteria on the cultivation-dependent and cultivationindependent bacterial communities in the root endosphere and rhizosphere soil of the INTRODUCTION Due to mining and heavy metals from sewage irrigation, heavy metal-contaminated cultivated land accounts for approximately one-fifth of the total cultivated land in China. This large-scale heavy metal pollution of farmland has serious consequences for food security in China and necessitates remediation and treatment (Li et al., 2014;Zhao et al., 2015;Duan et al., 2016). Phytoremediation has become an important area of research within soil heavy metal pollution remediation because it does not produce secondary pollution, is a simple operation, and incurs low investment costs (Cunningham et al., 1995;Liu et al., 2018).
During the remediation of heavy metal-contaminated soil, plants grow slowly and have reduced biomass due to heavy metal stress, thus limiting their remediation efficiency (Ullah et al., 2015). There are many types of microorganisms in soil, which are present in large quantities, have high biological activity, and play important roles in the growth of remediation plants in heavy metal-contaminated soils and in the geochemical cycle of heavy metals (Teng et al., 2015;Chen et al., 2017;Jing and Kjellerup, 2018). Bacteria in soil can improve plant nutrition through phosphorus solubilization and nitrogen fixation and through the secretion of plant hormones [indole-3-acetic acid (IAA), etc.], siderophores, and specific enzymes [1-aminocyclopropane-1carboxylate (ACC) deaminase, etc.], thus promoting the growth of remediation plants and the enrichment of heavy metals under stress as well as improving the heavy metal phytoremediation efficiency in soil. Due to their complementation ability, plantmicrobe-combined remediation is currently a research hotspot (Glick, 2003;Ma et al., 2016;Ashraf et al., 2017). To date, researchers have inoculated hyperaccumulator plants such as Solanum nigrum (Luo et al., 2011), Thlaspi caerulescens (Whiting et al., 2001), and Sedum plumbizincicola , energy plants such as Zea mays (Sheng et al., 2012) and Napier grass (Wiangkham and Prapagdee, 2018), and high-biomass plants such as poplar  with plant growth-promoting bacteria (PGPB) to investigate their effects on plant growth and their heavy metal remediation efficiency in soils. However, most investigations of combined plant-PGPB remediation for soil heavy metal pollution have been performed under simple or controllable conditions such as potted plants or greenhouses. Field trials with more complicated environmental conditions have rarely been reported, while field trials are a necessary stage for combined plant-PGPB remediation technology to progress from the laboratory to practical applications, and the related work must be continued (Zhuang et al., 2007;Dary et al., 2010;Kong and Glick, 2017;Ju et al., 2019). Prapagdee and Khonsue (2015) showed that the inoculation of cadmium-resistant Ralstonia sp. TISTR 2219 and Arthrobacter sp. TISTR 2220 bacteria under field conditions increased cadmium accumulation in the roots, above-ground tissues, and whole plants of Ocimum gratissimum. The "in situ" remediation experiment conducted by Dary et al. (2010) showed that the inoculation of Lupinus luteus with metal-resistant PGPB increased the biomass and reduced the metal accumulation.
Rape is a cruciferous Brassica plant, for which many species or genotypes have strong heavy metal accumulation characteristics (Rizwan et al., 2018). Research examining the combined remediation of heavy metal pollution in soil using rape as a remediation plant inoculated with PGPB has been performed under potted or greenhouse conditions, but in situ field trials for related research have rarely been reported (Farwell et al., 2006;Sheng and Xia, 2006;Chen et al., 2013;Dąbrowska et al., 2017;Pan et al., 2017). Therefore, the primary objectives of this study were (1) to evaluate the effect of PGPB inoculation on the growth of rape and its enrichment of heavy metals in the field around a region characterized by heavy metal mine tailings, (2) to determine the effect of PGPB on the antioxidant activity of rape during the enrichment of soil heavy metals, and (3) to determine the effect of PGPB inoculation on the composition of the bacterial community in the rape rhizosphere and endosphere. Through the above research, we expected to perform a preliminary exploration of the PGPB mechanism that improves plant enrichment of heavy metals under field conditions from the perspectives of plant physiological indicators and microbial communities.
The field experiment site is farmland near Nanjing Jiuhuashan Cu mining, on Funiu mountain in Tangshan town (32 • 04'N; 119 • 05'E), Jiangning District of Nanjing, which was polluted by Cu mining. The soil characteristics include a pH of 6.68, organic matter content of 3.04 g kg −1 , cation exchange capacity (CEC) of 14.5 cmol kg −1 , total nitrogen (N) of 1.41 g kg −1 , phosphorus (P) of 13.4 mg kg −1 , and potassium (K) of 85.7 mg kg −1 , along with heavy metal (Cu, Zn, Pb, and Mn) contents of 1068.25, 133.00, 34.50, and 534.75 mg kg −1 . With reference to Soil Environmental Quality Standard Two (GB 15618-1995), the Cu pollution at the field experiment site exceeded grade III for agricultural land (400 mg kg −1 ).

Introduction of Strains J62, JYC17, and Y1-3-9 and Plant Growth
The field experiments were performed during the season between September and November in 2012. The fields were divided into plots for rape (variety Qinyou-7) planting. The field experiments were divided into four treatments, namely, J62 inoculation, Y1-3-9 inoculation, JYC17 inoculation, and an uninoculated control. Each plot was 52 m 2 (13 m × 4 m) in area, with plants growing in a row spacing of 0.5 m × 0.5 m (Supplementary Figure S1A). The field plot experiments were conducted using a randomized arrangement with three replications. For the inoculation, strains J62, JYC17, and Y1-3-9 were grown in LB medium for 18 h at 28 • C with continuous shaking at 200 rpm, and the cells were collected by centrifugation at 6,000 rpm for 10 min, washed, and recentrifuged in sterile distilled water to obtain a bacteria inoculum of approximately 10 8 cfu mL −1 . The bacterial suspensions (10 mL plant −1 ) were sprayed on the rape rhizosphere. For the uninoculated control, an equal volume of sterile water was added.
Fifty days after the inoculation treatment, five plants were harvested at random from the central parts of each plot (Supplementary Figure S1B). Rhizosphere soil was obtained by first gently shaking off the loosely bound soil, while the rhizosphere soil adhering to the root system was isolated by more vigorous shaking or by hand. The above-ground tissues and roots were separated and washed extensively, first in several changes of 0.01 M EDTA and then in distilled water to remove any non-specifically bound heavy metals, and then they were dried at 80 • C for 2 days before the root and above-ground tissue dry weights were determined (Supplementary Figure S1C). The root and above-ground tissues were digested in a mixture of concentrated HNO 3 and HClO 4 (4:1, v/v), and the Cu contents of the samples were determined with an inductively coupledplasma optical emission spectrometer (ICP-OES) (Optima 2100 DV, Perkin Elmer). The water-soluble and NH 4 OAc-extractable Cu concentrations in the rhizosphere soils of the plants were determined by ICP-OES, and the pH of the soil (1:1 w/v water) was determined with a pH meter.

Determination of Antioxidant Enzymes and Thiobarbituric Acid-Reactive Substances (TBARS)
For the enzyme extraction, fresh leaves (0.5 g) were homogenized in 10 mL of ice-cold potassium phosphate buffer (pH 7.0) in an ice bath by grinding using a mortar and pestle. The homogenate was centrifuged at 12,000 g for 20 min at 4 • C. The supernatant was stored at 4 • C and used to determine the various antioxidant enzymes. The superoxide dismutase (SOD) activity was measured through the photoreduction of nitro blue tetrazolium chloride (NBT) (Dhindsa and Matowe, 1981). The peroxidase (POD) activity was measured according to the method by Rao et al. (1996). The glutathione (GSH) was measured according to Griffith (1980), and the ascorbic acid (ASA) was measured according to Arakawa et al. (1981). The protein content was determined according to the Bradford (1976) method with bovine serum albumin as the standard. The TBARS was measured as described by Jiang and Zhang (2001).

DNA Extracted From Biomass Was Accumulated on an Agar Plate (Cultivation-Dependent) and Extracted Directly From the Endosphere and Rhizosphere Samples (Cultivation-Independent)
Culturable endophytic and rhizosphere bacteria were isolated by traditional plate culture methods. The plant roots were surface-sterilized by sequential immersion in 75% (v/v) ethanol for 2 min and 1% mercuric chloride for 1 min, they were ground with a mortar and pestle in the presence of 5 ml of sterile distilled water, and then they were spread on plates containing 1/5-strength LB medium for 72 h at 28 • C to isolate the endophytic bacteria. Samples of rhizosphere soil weighing 1 g were taken from each treatment, homogenized in 10 mL of 0.85% saline, and serially diluted (10-fold) in the same container, and the aliquots (100 µL) were spread on 1/5 LB medium and incubated for 72 h at 28 • C. DNA was extracted from the bacterial biomass on the plates by cetyltrimethylammonium bromide (CTAB) method (Ellis et al., 2003), and the results were considered "cultivationdependent" samples.
Endophytic bacteria from the plant roots was extracted as described by Sun et al. (2010). The plant roots were surfacesterilized as described above for the isolation of culturable endophytic bacteria, they were ground in a sterilized mortar with 5 mL sodium phosphate buffer, and then they were transferred to tubes that were shaken for 1 h to dislodge the bacterial cells from inside the plant tissue. The bacterial cells were collected by centrifugation at 12000 g for 10 min and resuspended in 550 µL of TE buffer. The total endophytic bacterial DNA was extracted as described by Araújo et al. (2002). The rhizosphere bacterial DNA was extracted based on a modification of a method by Zhou et al. (1996). The resulting extracts were considered "endosphere" and "rhizosphere" samples.

PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Analyses
The DGGE primers GC-341F and 534R were used to amplify the V3 hypervariable region of 16S rRNA genes directly from the DNA samples (Muyzer et al., 1993). The PCRs were performed in a PTC-200 DNA Engine Cycler (Bio-Rad, United States) using the amplification program and reaction conditions described by Chen et al. (2013). For the DGGE analysis, the PCR products generated from each sample were separated on an 8% acrylamide gel with a linear denaturant gradient ranging from 45 to 75% using a DCode TM Universal Mutation Detection System from BIO-RAD, United States. The DGGE was performed using 20 µL of the PCR product in 1× TAE buffer at 60 • C, 200 V for 10 min, then 85 V for 12 h. The gels were stained with SYBR Green I (Generay Biotech Co., Ltd., Shanghai, China) in 1× TAE for 30 min, and the gels were scanned with a gel photo GS-800 system (Bio-Rad, United States).
The bands of interest were excised and eluted with 50 µL of ddH 2 O for 24 h at 4 • C. The resulting solution (1 µL) was used as the target DNA for a subsequent PCR amplification with primers 341F and 534R, and the PCR products were cloned into the pGEM-T Easy vector as described by the manufacturer. The clones were sequenced, and these short fragments were subjected to BLAST-assisted searches of the NCBI database; the closest match of known phylogenetic affiliation was used to assign the bands to taxonomic groups.

Statistical and Cluster Analyses
The microbial community banding profiles on the DGGE gels were analyzed using a Quantity One software package (Bio-Rad Laboratories, Inc., Hercules, United States). The plant growth parameter data were analyzed using a Student-Newman-Keuls test at a significance level of P < 0.05. All the statistics were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, United States).

Effects of Each Strain on the Biomass and Cu Enrichment in Rape
The remediation rape plants grew well in the Cu-contaminated farmland, and the inoculation treatment promoted plant growth. The promotion of above-ground biomass (dry weight) was more obvious than the promotion of root growth. Compared with the plants without inoculation treatment, the above-ground biomass after the inoculation treatment increased by 27.3-59.5%, and the root biomass increased by 8.6-67.2%. The increases in the aboveground biomass of rape inoculated with strains JYC17 and J62 were significantly different from those for the control without inoculation treatment (P < 0.05) (Figure 1). Simultaneously, the inoculation treatment affected the Cu concentration in the rape to varying degrees. The JYC17 and Y1-3-9 increased the Cu concentration in the roots of the rape, with values of 42.63 and 40.14 mg kg −1 , respectively, demonstrating increases of 41.48 and 34.13%, compared with 30.13 mg kg −1 in the control group, for which the former difference was statistically significant (P < 0.05). Y1-3-9 and J62 increased the Cu concentrations in the above-ground part of the rape by 10.27 and 25.56%, respectively (Figure 2). The translocation factor (TF) indicates the capacity of the plants to translocate heavy metals from the roots to the above-ground tissues, which is expressed as the ratio of the heavy metal concentration in the above-ground tissues/the heavy metal concentration in the roots. The experimental results showed that the TFs of plants inoculated with JYC17, Y1-3-9, and J62 were 0.33, 0.41, and 0.85, respectively. Strain J62 significantly increased the TF by 70.68% compared with the control (P < 0.05). Although the tested bacteria showed different effects on the Cu concentration in different parts of the rape, the inoculation treatment increased the above-ground biomass of the plant, such that the total amount of Cu enrichment in the rape showed different degrees of improvement compared with the control. The total amounts of Cu enrichment in the roots of each rape plant inoculated with JYC17 and Y1-3-9 were 1193.90 and 727.02 µg plant −1 , respectively, demonstrating 210.2 and 88.9% significantly higher enrichment compared with the control (P < 0.05). The total amounts of Cu enrichment in the above-ground part of each rape plant inoculated with JYC17, Y1-3-9, and J62 were 1296.87, 1232.14, and 1696.69 µg plant −1 , respectively, demonstrating increases of 63.4, 55.3, and 113.8% compared with the control value of 793.47 µg plant −1 . The total Cu enrichment in whole plants inoculated with JYC17, Y1-3-9, and J62 increased by 113.4, 66.3, and 67.9%, respectively (Figure 3).

Effect of Strain Inoculation on the Soil Bioavailability of Heavy Metals
The bioavailability of heavy metals in the plant rhizosphere is an important factor limiting the efficiency of phytoremediation. The  effects of different treatments on the forms of Cu in the rape rhizosphere soil was determined, and the results are shown in Table 1. Accordingly, the inoculation using the tested bacteria changed the forms of Cu in the soil. Strains Y1-3-9 and J62 increased the levels of water-soluble Cu in the rape rhizosphere soil to 1.12 and 0.87 mg kg −1 , respectively, demonstrating increases of 41.77 and 10.13% compared with the control. The contents of NH 4 OAc-extracted and DTPA-extracted Cu after the inoculation treatment were lower than those in the control group, to different degrees. The inoculation showed little effect on the pH of the rhizosphere soil.

The Effect of Strain Inoculation on the Antioxidant Activities in Rape Leaves
Under an environment of heavy metal stress, non-enzymatic substances composed of ASA and GSH can facilitate a plant's elimination of peroxidation. According to the ASA content measurements shown in Figure 4, the inoculation using the tested bacteria increased the ASA contents in the rape leaves. The ASA content increased from 456.68 mg kg −1 fresh weight (FW) in the control to 873.26 mg kg −1 FW in the plants inoculated with JYC17 (91.22% increase), 640.47 mg kg −1 FW in the plants inoculated with Y1-3-9 (40.24% increase), and 913.37 mg kg −1 FW in the plants inoculated with J62 (80.49% increase). Similar to the ASA results, inoculation using the tested bacteria increased the GSH contents in the rape leaves. The GSH content increased from 373.47 mg kg −1 FW in the control to 415.15 mg kg −1 FW in the plants inoculated with JYC17 (11.16% increase), 439.46 mg kg −1 FW in the plants inoculated with Y1-3-9 (17.67% increase), and 410.41 mg kg −1 FW in the plants inoculated with J62 (9.89% increase).
The TBARS content in plants is a physiological indicator of plant stress. The increase in the TBARS content in a plant indicates that the plant is under a certain degree of stress. The TBARS contents in rape leaves inoculated with JYC17, Y1-3-9, and J62 were decreased to different extents compared with the control (10.16, 10.58, and 21.88%, respectively). Similar to the effect of TBARS in plants, the SOD and POD activities in plants exposed to adverse conditions such as heavy metal stress can be enhanced, and the reactive oxygen species (ROS) caused by the stress in the plants can be scavenged. Therefore, they can also be used as a physiological indicator of plant stress. According to the SOD and POD activity assay shown in Figure 4, JYC17, Y1-3-9, and J62 inoculation reduced the POD activity in the rape leaves by 9.39, 4.55, and 17.80%, respectively. Concomitantly, inoculating with strain J62 reduced Means within the same column followed by the same letter are not significantly different at P < 0.05, based on the Student-Newman-Keuls test.
the SOD activity in the rape leaves by 5.99%; the remaining two groups showed little change compared with the control. Combined with the changing TBARS pattern in the plants, the results suggested that inoculating with the tested bacteria reduced the physiological indicator of plant stress in the Cu-polluted farmland soil for rape.

Composition of the Cultivation-Dependent and Cultivation-Independent Bacterial Community on the Rape Root Endosphere and Rhizosphere Soil
The band mobility of each sample in the DGGE profile was analyzed using Quantity One analysis software. A total of 23 bands from the cultivation-dependent profile and 43 bands from the culture-independent profile were cut and recovered (Figure 5). These bands were sequenced, and a homology alignment was performed with the known sequences in GenBank using BLAST software. The application of accession number HQ603005-HQ603051 was submitted to the GenBank database. Based on the sequencing results for the bands, the composition of the cultivation-dependent bacterial community in the rape root endosphere and its rhizosphere soil in the heavy metalcontaminated farmland near the Cu mine was as follows: Gammaproteobacteria, Firmicutes, Betaproteobacteria, and Actinobacteria, with 10, 7, 3, and 1 bands and proportions of the 21 sequenced bands at 47.6, 33.3, 14.3, and 4.7%, respectively ( Figure 5A and Supplementary Table S1). Bacillus spp. in Gammaproteobacteria and Pseudomonas spp. in Firmicutes were the dominant populations among the cultivation-dependent bacteria, accounting for 47.6 and 28.6% of the total numbers of sequenced bands, respectively. The bands in the cultivation-independent DGGE profile of the directly extracted DNA samples were sequenced, and they primarily consisted of seven bacterial populations, including Alphaproteobacteria (1 band, 3.8%), Betaproteobacteria (2 bands, 7.7%), Gammaproteobacteria (5 bands, 19.2%), Firmicutes (2 bands, 7.7%), Actinobacteria (2 bands, 7.7%), Bacteroidetes (3 bands, 11.5%), and uncultured bacteria (11 bands, 42.3%) ( Figure 5B and Supplementary Table S1). Rhodanobacter spp. and Pseudomonas spp. in Gammaproteobacteria and Burkholderia sp., Janthinobacterium sp. and Achromobacter sp. in Bacteroidetes were the dominant populations in the endosphere and rhizosphere samples.

Effects of the Tested Bacteria on the Bacterial Communities in the Endosphere and Rhizosphere
The effects of the tested bacteria on the number of cultivationdependent bacteria in the rape rhizosphere and endosphere were determined. The results showed that the total number of bacteria in the rhizosphere of the tested plants was 10 6 cfu g −1 soil and that the total number of bacteria in the roots was approximately 10 4 cfu g −1 fresh weight. Inoculating with Y1-3-9 and JYC17 increased the number of bacteria in the rhizosphere and the roots. Simultaneously, the sequencing results for the DGGE bands showed that the sequence information for bands A15, A16, and A23 corresponded to the test bacteria Y1-3-9, J62, and JYC17, respectively, with a similarity of 100%. Based on the DGGE sequencing results and the counts of cultivation-dependent bacteria, the tested bacteria colonized the rape endosphere and rhizosphere under field conditions ( Table 1, Supplementary  Table S1, and Figure 5A).
To investigate the effects of the tested bacteria on the bacterial community structure of the rape endosphere and its rhizosphere, the DGGE profile was subjected to a canonical correspondence analysis (CCA), and the results are shown in Figure 6. Figure 6A shows the CCA of the cultivation-dependent DGGE profile. The cultivation-dependent bacterial community in the endosphere (E-CK) and the cultivation-dependent bacterial community in the rhizosphere (R-CK) in the control group without strain inoculation was separated through axis 1 (36.9%), indicating that the structure of the cultivation-dependent bacterial communities in the root endosphere was significantly different from that of the cultivation-dependent bacterial communities in the rhizosphere soil. Following inoculation with the tested bacteria, the endosphere cultivation-dependent bacteria community and rhizosphere cultivation-dependent bacteria community were clustered together above axis 2 (18.5%) of the CCA profile, in which the endosphere cultivation-dependent bacteria (E-J62) and rhizosphere cultivation-dependent bacteria (R-J62) for strain J62 and the endosphere cultivation-dependent bacteria (E-Y1-3-9) and rhizosphere cultivation-dependent bacteria (R-Y1-3-9) for strain Y1-3-9 were clustered together and separated from those of the control group (E-CK and R-CK). This finding indicates that the test bacteria J62 and Y1-3-9 affected the structure of the cultivation-dependent bacterial communities in the rape endosphere and its rhizosphere and had similar community structures. Figure 6B shows the CCA of the cultivation-independent DGGE profile. Axis 1 divided the endosphere bacteria and rhizosphere bacteria into two parts at 31.0%, indicating that different sample sources (the rape endosphere and rhizosphere) are the most important factors determining the structure of the bacterial communities. Based on the CCA, the community structure in the control group at day 0 for the endosphere bacteria (E-CK0) and rhizosphere bacteria (R-CK0) was separated from the community structure at day 50 for the endosphere bacteria (E-CK) and rhizosphere bacteria (R-CK), indicating that the bacterial community changed over time. The E-Y1-3-9, E-JYC17, and E-J62 for the rape endosphere following inoculation were separated from each other in the CCA profile, while E-Y1-3-9 was closer to the E-CK of the control group, indicating that inoculation with the tested bacteria affected the bacterial communities of the rape root endosphere and that the degrees of influence from the different strains were different. Similarly, the R-Y1-3-9, R-JYC17, and R-J62 for the rhizosphere after inoculation treatment were clustered together in the CCA profile and separated from the R-CK , 50-day inoculation with Y1-3-9 (E-Y1-3-9), and 50-day inoculation with J62 (E-J62); lanes 5 to 8, rhizosphere samples from the 50-day uninoculated control (R-CK), the 50-day inoculation with JYC17 (R-JYC17), the 50-day inoculation with Y1-3-9 (R-Y1-3-9), and the 50-day inoculation with J62 (R-J62). (B) Lanes 1 to 5, endosphere samples from the 0-day uninoculated control (E-CK0), the 50-day uninoculated control (E-CK), the 50-day inoculation with JYC17 (E-JYC17), the 50-day inoculation with Y1-3-9 (E-Y1-3-9), and the 50-day inoculation with J62 (E-J62); lanes 6 to 10, rhizoplane samples from the 0-day uninoculated control (R-CK1), the 50-day uninoculated control (R-CK2), the 50-day inoculation with JYC17 (R-JYC17), the 50-day inoculation with Y1-3-9 (R-Y1-3-9), and the 50-day inoculation with J62 (R-J62). M, marker lane consisting of 16S rRNA gene fragments from J62, JYC17 and Y1-3-9. The bands in the DGGE profiles were excised and sequenced, and their given numbers correspond to the list in Supplementary Table S1. of the control group, indicating that the inoculation with the tested bacteria affected the bacterial communities of the rape rhizosphere soil.
A bacterial community similarity clustering analysis was performed on the DGGE profile according to the unweighted pair group method with the arithmetic mean (UPGMA) algorithm, which was similar to the CCA results. The cultivation-dependent bacteria in the endosphere samples were clustered at a similarity level of 0.39 and were separated from the rhizosphere samples, indicating that the different sample source was the most important factor affecting the composition of the cultivation-dependent bacterial communities (Supplementary Figure S2). Concurrently, the inoculated and non-inoculated samples were separated from each other in the cluster analysis profile, indicating that inoculating with the tested bacteria affected the composition of the cultivationdependent bacterial communities in the rape endosphere and rhizosphere. Similarly, the cultivation-independent bacteria in the rhizosphere samples were clustered at a similarity level of 0.43 and were separated from the rhizosphere samples. Simultaneously, the inoculated and non-inoculated samples were separated from one another in the cluster analysis profile.

DISCUSSION
During the combined plant-microbe remediation of heavy metal pollution in soil, PGPB can regulate the physiological processes of plants and reduce the stress from heavy metals on plants; simultaneously, the strains can activate insoluble heavy metals and phosphorus nutrients in the soil by producing iron carriers and organic acids. These bacteria can change the bacterial community structure of the rhizosphere soil and its endosphere and improve the biomass of the remediation plant, thus improving the efficiency of phytoremediation (Teng et al., 2015;Ma et al., 2016;Ashraf et al., 2017). However, microbes have a strong dependence on the environment, and changes in environmental conditions can modulate the effects of microbes. To date, studies related to the plant-PGPB remediation of heavy metal pollution in soil have primarily been performed under conditions that are relatively easy to control, such as in pots or in a greenhouse. Few studies have examined the effects of these strains under complex field conditions, limiting the practical application of combined plant-PGPB remediation (Zhuang et al., 2007;Dary et al., 2010;Kong and Glick, 2017). The remediation field trials conducted by Prapagdee and Khonsue (2015) and Dary et al. (2010) showed that the inoculation of metal-resistant FIGURE 6 | Canonical correspondence analysis (CCA) of DGGE bacterial community profiles from panel (A) DNA extracted from biomass accumulated on an agar plate (cultivation-dependent, Figure 5A) and panel (B) DNA extracted directly from endosphere and rhizosphere samples (cultivation-independent, Figure 5B). The values on the axes indicate the total variation percentages explained by each axis. (A) Endosphere samples from the 50-day uninoculated control (E-CK), 50-day inoculation with JYC17 (E-JYC17), 50-day inoculation with Y1-3-9 (E-Y1-3-9), and 50-day inoculation with J62 (E-J62); rhizosphere samples from the 50-day uninoculated control (R-CK), the 50-day inoculation with JYC17 (R-JYC17), the 50-day inoculation with Y1-3-9 (R-Y1-3-9), and the 50-day inoculation with J62 (R-J62). The digital numbers represent 23 bands from the cultivation-dependent profile ( Figure 5A). (B) Endosphere samples from the 0-day uninoculated control (E-CK0), the 50-day uninoculated control (E-CK), the 50-day inoculation with JYC17 (E-JYC17), the 50-day inoculation with Y1-3-9 (E-Y1-3-9), and the 50-day inoculation with J62 (E-J62); rhizoplane samples from the 0-day uninoculated control (R-CK1), the 50-day uninoculated control (R-CK2), the 50-day inoculation with JYC17 (R-JYC17), the 50-day inoculation with Y1-3-9 (R-Y1-3-9), and the 50-day inoculation with J62 (R-J62). The digital numbers represent 43 bands from the culture-independent profile ( Figure 5B).
PGPB improved the biomass of the remediation plant L. luteus and achieved the purpose of increasing or decreasing the metal accumulation in O. gratissimum and L. luteus. The experimental results showed that the combined plant-PGPB remediation of heavy metal pollution in soil has a certain practical value under field conditions. In our previous pot trials, metal-resistant PGPB J62 and Y1-3-9 reduced the Cd stress in rape and increased the total amount of Cd uptake by the plant. The cumulative amount was increased from 6.70 to 40.60% compared with the corresponding control (Chen et al., 2013). The results of the combined remediation field trials showed that inoculating with the tested bacteria promoted rape growth, increased the dry weight of the above-ground tissues and roots, affected the Cu concentration in the rape and increased the total amount of Cu uptake. The total Cu uptake in whole plants inoculated with JYC17, Y1-3-9, and J62 was increased by 113.38, 66.26, and 67.91%, respectively. PGPB can directly or indirectly dissolve insoluble heavy metals through various metabolic activities (Sessitsch et al., 2013). Strains Y1-3-9 and J62 can activate watersoluble Cd in the soil in pot experiments (Chen et al., 2013). Similarly, strains Y1-3-9 and J62 can increase the contents of water-soluble Cu in rape rhizosphere soil under field conditions. The above results indicate that strains JYC17, Y1-3-9, and J62 are effective at promoting the phytoremediation of heavy metal pollution in soil under field conditions and that the rape remediation system and strains Y1-3-9 and J62 have good adaptability, which can be applied to different conditions (potted plants and fields) for different heavy metal types (Cu and Cd pollution), showing a certain application value. However, the current experiments were conducted only for a single rape crop in a single soil contaminated with heavy metals. Experimental research to account for different crops, soil types, and growing environments is needed to investigate the combined remediation of heavy metal pollution in the soil using strains JYC17, Y1-3-9, and J62 under field conditions. Heavy metals in the soil often cause direct or indirect damage, inhibit the growth of remediation plants, and affect the efficiency of phytoremediation. Studies have shown that PGPB can alleviate oxidative stress and reduced chlorophyll synthesis, impact other physiological and biochemical indicators caused by heavy metals, and improve the adaptability of remediation plants to heavy metal pollution (Tak, 2015;Pan et al., 2016;Chiboub et al., 2018;Rizvi and Khan, 2018). Saleem et al. (2018) found that Pbtolerant PGPB increased the biomass and Pb uptake of sunflower remediation plants in pot experiments. Simultaneously, the indicators of physiological and antioxidant activities showed that PGPB increased the contents of chlorophyll "a, " chlorophyll "b, " carotenoids, ascorbate peroxidase, catalase, superoxide dismutase, glutathione reductase, and proline in sunflowers that were exposed to Pb contamination. These indicators play an important role in improving the photosynthetic activities and alleviating the oxidative stress caused by heavy metals. Similarly, this experiment determined the effects of inoculating the rape with PGPB on the antioxidant indicators under field conditions. The results showed that PGPB inoculation increased the contents of the non-enzymatic antioxidants ASA and GSH by 40.24-91.22% and 9.89-17.67%, respectively. Studies have shown that ASA and GSH are the most important antioxidants in plants, and they play important roles in scavenging the toxic ROS caused by heavy metal stress. The increased contents of these compounds help plants to tolerate various stresses. This result is similar to the findings of Pan et al. (2016) and Islam et al. (2014), who showed that inoculation by the tested bacteria increased the GSH and ASA contents and alleviated the oxidative stress caused by heavy metals. The TBARS, SOD, and POD contents in plants reflect the changes in ROS and hydrogen peroxide free radicals in plant tissues, so they can be used as indicative physiological indicators of plant stress. The TBARS and POD contents in the rape leaves treated with JYC17, Y1-3-9, and J62 were decreased to different degrees compared with the control, indicating that the oxidative stress caused by heavy metals was alleviated in the rape after the inoculation treatment.
Microorganisms are widely distributed in the plant rhizosphere and endosphere, and they play important roles in the phytoremediation of heavy metal-contaminated soil (Ashraf et al., 2017). PCR-DGGE is a modern molecular biology technology for studying microbial communities. It is widely used in microbial ecology investigations in different ecological environments such as plant samples (de Melo Pereira et al., 2012;Chen et al., 2013) and soil (Che et al., 2015;Orlewska et al., 2018). Rhizosphere and endosphere colonizations of PGPB in plants are the primary conditions for its effect. Because of its technical sensitivity, PCR-DGGE technology has been used to study strain colonization in soils or plants (Andreote et al., 2009;Chen et al., 2013;Rilling et al., 2019). Using the PCR products of strains JYC17, Y1-3-9, and J62 as the DGGE markers, and based on the positions of the corresponding bands on the cultivation-dependent and cultivation-independent DGGE profiles and the sequencing information for bands A15, A16, and A23 (Supplementary Table S1 and Figure 5), it was judged that the tested bacteria colonized the rape roots and rhizosphere under field conditions, consistent with the colonization results obtained for strains J62 and Y1-3-9 in the rape rhizosphere and endosphere during previous potting trials (Chen et al., 2013). Studies concerning the interaction of PGPB with microorganisms in soil or the plant endosphere during combined plant-microbe remediation, as well as the impact on the composition of the microbial community in soil, have also been conducted (Jeong et al., 2013;Marques et al., 2013;Liu et al., 2015). Liu et al. (2015) showed that the inoculation of heavy metal-tolerant PGPB during the remediation of Cd-contaminated soil by S. plumbizincicola reduced the diversity of the rhizosphere microbial community. Cultivation-dependent bacteria play an important role in the microbial community and its activity. In traditional studies that have examined the composition of cultivation-dependent bacterial communities, cultivation-dependent bacteria are generally first enriched and cultured in a medium, followed by separation, purification, and identification, which is time-consuming and laborious. Based on the results reported by Ellis et al. (2003), the total DNA from bacteria growing on a plate were extracted, and a PCR-DGGE analysis was performed to analyze the effects of the tested bacteria quickly and accurately on the composition of the microbial community of the cultivation-dependent bacteria in the rape rhizosphere soil and root endosphere (Supplementary Table S1 and Figure 5). Using CCA and similarity clustering analyses of the cultivation-dependent DGGE profile, it was concluded that the tested bacteria inoculated with JYC17, Y1-3-9, and J62 under field conditions affected the composition of the cultivation-dependent bacterial communities in the rape root endosphere and rhizosphere soil and that the effects of the different strains were different. These results were consistent with previous findings demonstrating that the cultivation-dependent bacterial community composition of the rape rhizosphere and endosphere was affected by strains J62 and Y1-3-9 under potting conditions (Chen et al., 2013). Similar to the change in the cultivation-dependent bacterial community composition, the cultivation-independent DGGE profile results showed a significant difference between the rape root and rhizosphere. Inoculation with the test bacteria affected the composition of the bacterial communities in the endosphere and rhizosphere to different extents. These results are similar to previous findings showing that inoculation by plant growth-promoting rhizobacteria can alter the bacterial community composition of the plant rhizosphere or endosphere (Andreote et al., 2010;Chen et al., 2013;Liu et al., 2015). These changes in the community composition, especially the colonization of the test bacteria JYC17, Y1-3-9, and J62 with good growth-promoting abilities in the rape roots and rhizosphere, significantly enhanced the proportion of growth-promoting bacteria among the total cultivation-dependent bacteria, which may play an important role in the heavy metal stress tolerance of rape.

CONCLUSION
The results of the in situ remediation experiments on rapepromoting bacteria in the Cu-contaminated farmland around heavy metal mine tailings showed that metal-resistant PGPB increased the rape biomass and the total amount of Cu uptake, thus enhancing the Cu enrichment efficiency of rape. Simultaneously, the mechanism of this effect was preliminarily analyzed from the perspectives of the plant physiology and the microbial community structure. The results showed that the test bacteria JYC17, Y1-3-9, and J62 had good growth-promoting abilities, colonized the rape rhizosphere and endosphere, and altered the bacterial community composition of the rape rhizosphere and endosphere. Concurrently, they increased the ASA and GSH contents in the rape and reduced the oxidative stress caused by heavy metals. The above results indicate that the plant-promoting bacteria JYC17, Y1-3-9, and J62 are effective at promoting the phytoremediation of heavy metal pollution in soil under field conditions, and they could have value for use in specific applications.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
Z-JC designed the experiments and contributed to writing the manuscript. X-MR and S-JG performed the experiments and analyzed the data. HH and YC performed the field sampling. WT and EC measured the heavy metals concentrations in the rape. B-LL and Y-YL reviewed the manuscript.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Agricultural And Food Sciences
Biology

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

text:
Comparative RNA-Seq Analysis of Differentially Expressed Genes in the Epididymides of Yak and Cattleyak

Background: Cattleyak are the Fl hybrids between (♀) yak (Bos grunniens) and (♂) cattle (Bos taurus). Cattleyak exhibit higher capability in adaptability to a harsh environment and display much higher performances in production than the yak and cattle. The cattleyak, however, are females fertile but males sterile. All previous studies greatly focused on testes tissues to study the mechanism of male infer-tility in cattleyak. However, so far, no transcriptomic study has been conducted on the epididymides of yak and cattleyak. Objective: Our objective was to perform comparative transcriptome analysis between the epididymides of yak and cattleyak and predict the etiology of male infertility in cattleyak. Methods: We performed comparative transcriptome profiles analysis by mRNA sequencing in the epidi-dymides of yak and cattleyak. Results: In total 3008 differentially expressed genes (DEGs) were identified in cattleyak, out of which 1645 DEGs were up-regulated and 1363 DEGs were down-regulated. Thirteen DEGs were validated by quantitative real-time PCR. DEGs included certain genes that were associated with spermatozoal matura-tion, motility, male fertility, water and ion channels, and beta-defensins. LCN9, SPINT4, CES5A, CD52, CST11, SERPINA1, CTSK, FABP4, CCR5, GRIA2, ENTPD3, LOC523530 and DEFB129, DEFB128, DEFB127, DEFB126, DEFB124, DEFB122A, DEFB122, DEFB119 were all downregu-lated, whereas NRIP1 and TMEM212 among top 30 DEGs were upregulated. Furthermore, protein processing in endoplasmic reticulum pathway was ranked at top-listed three significantly enriched KEGG pathways that as a consequence of abnormal expression of ER-associated genes in the entire ER protein processing pathway might have been disrupted in male cattleyak which resulted in the down-regulation of several important genes. All the DEGs enriched in this pathway were downregulated ex-cept NEF. Conclusion: Taken together, our findings revealed that there were marked differences in the epididymal transcriptomic profiles of yak and cattleyak. The DEGs were involved in spermatozoal maturation, mo-tility, male fertility, water and ion channels, and beta-defensins. Abnormal expression of ER-associated genes in the entire ER protein processing pathway may have disrupted protein processing pathway in male cattleyak resulting in the downregulation of several important genes involved in sperm maturation, motility and defense.

INTRODUCTION
Cattleyak (CY) are the Fl hybrids between yak (YK) (♀) and cattle (CL) (♂). The cattleyak, also known as "khainag", are females fertile but males sterile. The backcrossing of F1 female cattleyak with either male yak or cattle respectively produces B1 backcross animal called "ortoom", that is also male sterile but female fertile. Moreover, backcrossing of B1 animal females either male yak or cattle produces B2 backcross animal called the "usanguzees", which are both male and female fertile [1]. Cattleyak display higher potential in *Address correspondence to this author at the School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, Sichuan, China; Tel: +86 08162419792; E-mail: caixin2323@126.com adapting to harsh environment and exhibit much higher performances in production such as economic traits, milk and meat production as compared to both yak and cattle. Cattleyak, therefore, has a great impact on the socio-economic development in the yak breeders in plateau regions, although how to make F1 male cattleyak fertile to utilize its hybrids is the key task in yak breeding [2].
Following spermatogenesis, spermatozoa formed in the testis are carried to the epididymis, where post-testicular maturation of the sperm cells occurs. Post-testicular maturation of spermatozoa mainly involves motility acquisition and the competence to undergo capacitation, leading to the ability to fertilize an egg [3,4]. The epididymis is functionally and structurally divided into three distinctive anatomical segments: the caput, the corpus and the cauda. Each segment possesses a distinct pattern of gene expression associated with physiological functions needed for the sperm maturation and motility [5][6][7]. The epididymis performs three major functions. The first main function is the transportation of sperm, which is accomplished by the smooth muscle cells contractions around the epididymal tube, and by constant movement and fluid secretions from the testis. The second main function is the maturation of spermatozoa. In the course of their passage through the epididymis, spermatozoa become fully mature and motile and get the ability to fertilize the oocyte. Maturation goes through a series of changes such as nuclear condensation and modifications in the proteins and lipids composition of the sperm plasma membrane. The third important function is the storage of spermatozoa, especially in the caudal epididymal segment [8,9]. During the maturation of spermatozoa, they undergo a series of molecular and biochemical changes as they pass through the epididymis. These changes are believed to be initiated by the transport of ions and the addition of proteins secreted in the epididymal duct lumen [8][9][10]. Genes expressed in the epididymis have a very important role in sperm maturation and motility; alterations in the expression levels of these epididymal specific genes may lead to infertility.
In recent studies, the certain number of genes have been identified to have putative roles in sperm maturation and motility. Examples of such genes are ROS proto-oncogene 1, receptor tyrosine kinase (Ros1) [11][12][13], glutathione peroxidase 5 (Gpx5) [14,15], murine epididymal retinoic acidbinding protein (Erabp), Acidic epididymal glycoprotein (Aeg1) [16,17], human epididymal proteins types 1-6 [18,19] and Bin1b [20], while many genes yet remain unknown [21,22]. Male Cattleyak infertility is one of the major hindrances in the yak breeding. Over the last few decades, researches have been conducted on the testes of YK and CY in order to investigate the mechanisms of infertility in male CY. No transcriptomic study, however, has been conducted on the epididymides of yak and cattleyak so far. In order to predict the etiology of infertility and to determine the differential gene expression patterns in the epididymal transcriptome of the YK and CY, we performed comparative mRNA sequencing (RNA-seq) analysis between the epididymal tissues of YK and CY. This study will enhance our understanding of the molecular mechanisms and differences in genes expression profiles between yak and cattleyak epididymal transcriptomes.

Sequencing and Assembly of Yak and Cattlyak Transcriptome
The cDNA libraries were generated using the mRNA from six epididymal tissue samples of yak and cattleyak (i.e., M1, M2, M3 P1, P2 and P3). Six biological samples of epididymis, i.e., a total of five samples were completed, three from the yak (control) and two replicates from cattleyak (treatment), were separately subjected to RNA-sequencing analysis. A total of 99.28 Gb of clean data were obtained from the five epididymal samples altogether, with Clean Data of 0.07Gb for each sample and 0.01% for Q30 ≥ bases and above. The efficiency of aligning reads with specified reference genome UMD3.1 of each sample ranged from 88.77% to 91.24% and matched to either unique or multiple genomic locations ( Table 1). In total, 109,496 unigenes including 21,681 new genes were mined in the bovine transcriptome. According to the databases sequence analysis of the newly discovered genes with Non-redundant protein sequences, NR [23], Swiss-Prot [24], Gene Ontology GO [25], Orthologous Groups COG [26] and Kyoto Encyclopedia of Genes and Genomes KEGG [27] databases using BLAST [28] total 18576 new genes were annotated in these databases. The replicates of yak and cattleyak repeated well with each other (r2=0.98~1.00) [29].

Comparative Analysis of Gene Expression
Comparative analysis of gene expression was performed for the yak and cattleyak epididymal tissues. In total 3008 unigenes, differential expressed genes were screened (Fig.  1). Three thousand and eight unigenes/DEGs, including 1363 down-regulated and 1645 up-regulated ones, were differentially expressed with false discovery rate (FDR) <0.05 and fold change (FC) ≥2 (Fig. 2). For the validation of RNA-seq data, we randomly selected 13 DEGs (APOL3, SYT4,  TMEM212, UBE2U, CES5A, COL11A2, CST11, Ooep,  CD52, DEFB124, LCN9, PRODH2 and MYT1L). The expression patterns of all thirteen differentially expressed genes (DEGs) were validated by quantitative real-time PCR (qPCR) and the results were consistent with those of the mRNA-Seq analysis ( Table 2). These results demonstrated that there is a significant difference in the number of differentially expressed genes in yak epididymal tissue compared to cattleyak epididymal tissue. Results of the functional   annotation indicated that all the DEGs had significant matches in the Nr database, and some of them also had annotation information in the Swiss-Prot, GO, KEGG and COG databases ( Table 3).
Based on the functional annotation obtained from GO enrichment analysis, the yak and cattleyak DEGs were separated into 54 functional groups, which belong to three main categories: biological processes (60 DEGs), cellular components (54 DEGs), and molecular functions (68 DEGs) (Fig.  3). In the biological process category, greater percentages of DEGs were involved in cell aggregation, cell killing, biological phase, growth, locomotion, localization, reproductive process, developmental process, multicellular organismal process, response to stimulus, multi-organism process and biogenesis compared to all unigenes of yak and cattleyak epididymal tissue. More proportions of DEGs in the cellular component category were localized to the collagen trimer, extracellular matrix part, synapse part, organelle part, synapse, membrane, membrane part and organelle. The DEGs in the molecular function category were more enriched in GO class of electron carrier activity, chemoattractant activity, antioxidant activity, transporter activity, enzyme regulator activity, receptor regulatory activity, channel regulator activity and nucleic acid binding transcription factor activity than compare to all unigenes.
Analysis of KEGG pathways was performed to determine the biological functions of the DEGs. Two hundred and sixty-nine (269) DEGs were allocated to two hundred and seventy-one (271) KEGG pathways (Fig. 4). The taste transduction pathway accounted for the highest number of DEGs (58 DEGs) followed by Huntington's disease, Protein processing in endoplasmic reticulum, Oxidative phosphorylation and Alzheimer's disease. In total, forty DEGs were allocated in protein processing in the endoplasmic reticulum pathway. ko04141 (Protein processing in the endoplasmic reticulum). All the DEGs enriched in ER protein processing pathway were downregulated except NEF which was upregulated in CY compare to YK. COG annotation grouped the DEGs into twenty-three COG functional classes. The top five COG classes with the highest number of DEGs included those involved in general function prediction only, posttranslational modification, protein turnover, chaperones, translation, ribosomal structure and biogenesis, signal transduction mechanisms, energy pro-duction and conversion and replication recombination and repair (Fig. 5).

DISCUSSION
The comparative transcriptome profiles analysis provided an outline of the transcriptomic diversity in yak and   cattleyak epididymal DEGs. Cattleyak exhibit higher capability in adaptability to a harsh environment and display much higher performances in production than the yak and cattle. Previous studies conducted on male cattleyak infertility have focused on testes whereas, so far, none of such studies have been conducted on the epididymal tissues in the male cattleyak to investigate the mechanism of infertility [35]. In this study, we performed comparative transcriptome analysis in the epididymides of yak and cattleyak to predict the etiology of male cattleyak infertility.
Given the fact that a number of genes could be differentially expressed in the epididymis of yak and cattleyak, transcriptomic comparison between the epididymes of yak and cattleyak is an effective approach to study or discover the key genes involved in different metabolic pathways that are specifically involved in sperm maturation and fertility.
Comparative analysis of gene expression between yak and cattleyak epididymal tissues was performed resulting in 3008 DEGs; 1363 down-regulated DEGs and 1645 upregulated DEGs. Our results demonstrated a significant difference in the number of DEGs between cattleyak and yak epididymal tissue. Functional annotation analysis performed for DEGs indicated that all the DEGs had significant matches in the Nr database while some DEGs additionally had annotation information in the Swiss-Prot, GO, KEGG and COG databases.
Interestingly, our results show that group of genes downregulated in the epididymis of infertile male cattleyak compared with yak encodes for proteins that are well known to play pivotal role in the host defense and spermatozoal maturation such as, DEFB129, DEFB128, DEFB127, DEFB126, DEFB124, DEFB122A, DEFB122, DEFB119 [36], CRISP1 [37] and SPINLW1 [38]. Since the ß -defensins were first identified in the epithelial cells of cattle airway as antimicrobials, more than 30 β-defensins genes have been described in bovines, human and other species [39][40][41]. Several are constantly expressed in the reproductive tract of male adult tissues and are particularly found to be abundant in the testis and epididymis [39, 42,43]. In some specific cases, their expression pattern is almost very abundant or restricted in the adult epididymal epithelium, where the majority of them depend on the β-defensin evaluated exhibit unique cell and region-specific expression patterns. It is also believed that Beta-defensins serve a very important role in both fertility and host defense [44][45][46]. Such as in innate immunity function of sperm coating proteins, inhibition of proteases may directly attack the sperm plasma membrane and antimicrobial activity. CRISP1 has been shown to play an important function in egg-sperm interaction and inhibition of the uptake of the ions, for instance, Ca2þ, is needed for spermatozoal capacitation [47]. Additionally, SPINLW1 binds with semeno-gelin in seminal plasma and on spermatozoa after ejaculation. It is believed that this protein complex is a part of a larger protein network on the sperm surface that acts as a protective shield in the female reproductive tract before capacitation [48].
Furthermore, another important group of genes, Lipocalins such as LCN9 (lipocalin 9), LCN6 (lipocalin 6), LCN12 (lipocalin 12) [49], which are all downregulated in the epididymis of infertile male cattleyak as compared to yak, consist of a family of proteins that play a role in the transportation of hydrophobic ligands, for instance, fatty acids retinoids and steroids to both the epithelium lining of the epididymis and the spermatozoa. LCN9 (-12.80 log2-fold change) which is reported to function in the transportation of luminal hydrophobic protein ligands to specific cells of epithelium lining of the epididymis [31] has an important but non-redundant function in male fertility. The functions of all the other epididymal Lipocalins, however, have not been so far well understood [50]. Interestingly, other genes having proteolytic activity, such as CTSK and ADAMDEC1, are also downregulated in epididymis of infertile male cattleyak compared with yak. CTSK is proteases that function in the degradation and processing of proteins and sperm maturation [51], while ADAMDEC1 is a proteolytically active metzincin metalloprotease [51]. Conversely, SPINT4 is an epididymis-specific protein having anti-proteolytic activity [52].
The remaining top 30 most DEGs were associated with CES5A (carboxylesterase 5A), an epididymis-specific gene which is crucial in sperm maturation and male fertility [53]. CD52 (CD52 molecule) is mainly secreted in the lumen of the epididymis and via the glycosyl-phosphatidylinositol (GPI) anchor. It is introduced into the sperm membrane during the passage of the spermatozoa through the epididymis. This is necessary for the maturation and mobility of sperm. Downregulation of CD52 is strongly associated with strong immobility and also lack of ability of sperm to penetrate the zona pellucida [54,55]. SERPINA1 (serpin family A member 1), lack of protein C inhibitor Serpina5 were found to cause infertility [56] while LOC523530 (nuclear RNA export factor 3), an epididymis-specific expression in principle cells regulates the expression of TGF-β3 in an mRNA export activity [57]. FABP4 (fatty acid-binding protein 4) associated with the stabilization of the sperm flagellum [58], CCR5 (C-C chemokine receptor type 5) essential for sperm motility, hyper-activation and acrosome reaction [18], GRIA2 (glutamate ionotropic receptor AMPA type subunit 2) functions in ion channels [31] and ENTPD3 (Ectonucleoside triphosphate) participates in smooth muscle contraction, steroidogenesis and spermatogenesis, and also regulates trans epithelial transport [59].
In the KEGG enrichment analysis of the DEGs, we selected the top four pathways obtained from the KEGG database analysis that were the taste transduction, Huntington's disease, protein processing in the endoplasmic reticulum, and the oxidative phosphorylation. Huntington's disease was identified as the top second in the KEGG database analysis. In the previous studies, it has been reported that ER stress is associated with many conformational diseases, such as Hun-tington's, Alzheimer's and Parkinson's disease [60][61][62], however, in our findings endoplasmic reticulum protein processing pathway was ranked at top three position in the KEGG database analysis, in which most of the enriched DEGs were downregulated (Figs. 4 and 5) and the oxidative phosphorylation was the top fourth enriched pathway in KEGG database analysis (Fig. 4).
The endoplasmic reticulum (ER) is membrane-bounded cytoplasmic organelle in the eukaryotic cells which serves many general functions such as synthesis and folding of proteins, lipids, and transportation of these synthesized proteins and lipids in vesicles to the Golgi apparatus. ER stress can be briefly defined as a disproportion between the protein folding ability of the ER and the protein load, consequential leads to misfolded proteins accumulation [63]. In these studies, it has been speculated that ER stress is involved in certain conformational diseases, such as Huntington's, Alzheimer's and Parkinson's disease [60][61][62]. Our findings showed that all the DEGs enriched in the "protein processing in endoplasmic reticulum pathway" process were downregulated except NEF in male cattleyak as compared to yak. We speculate that as a consequence of abnormal expression of ERassociated proteins, the entire ER protein processing pathway might have been disrupted in male cattleyak (Fig. 5).
ER chaperones comprise of molecular chaperones and folding enzymes localized in the ER, the function of which is to assist in the unfolding or folding and assembly of nascent proteins [64]. BIP (Binding protein) is a member of the HSP70 family, which is an important molecular chaperone by binding to the hydrophobic region of unfolded proteins and helps in folding [65]. Moreover, SEC63 and DNAJC3 are also molecular chaperones that have been reported as cochaperones which can regulate the function of BIP, downregulated in male cattleyak epididymal tissues. Furthermore, in our findings, numerous protein folding enzymes, such as OST (dolichyl-diphosphooligosaccharide protein glycosyltransferase) and PDI (protein disulfide isomerases) were reduced in the male cattleyak. These protein folding enzymes help in the oxidization of cysteine residues of nascent proteins and coordinate to make disulfide bonds. However, ERO1 (ER oxidoreduction) is involved in the reoxidation of reduced folding enzymes which was also down-regulated [66]. Additionally, the downregulation of XTP3B (XTP3transactivated gene B) and OS9 (osteosarcoma 9) which are very important ERAD (ER-associated degradation) complex components responsible for the identification of unfolded proteins, was downregulated in male cattleyak compare to yak [67-69]. Together, our findings suggest that downregulation of most of the DEGs involved in protein processing in the ER pathway may cooperate to cause the accumulation of some misfolded proteins in the ER, that leads to deficiency of vital proteins such as beta-defensins Lipocalins, etc. which are important in the sperm maturation, motility and host immunity, that might be the cause of infertility in male cattleyak. Additionally, the subsets of DEGS from this and other altered pathways could be further investigated first for technical validation and then for clinical applications.

Animals and Epididymal Sample Collection
Sample collection was carried out under license in accordance with the Guidelines for Care and Use of Laboratory Animals of China and all protocols were approved by the Institutional Review Board of Southwest University of Science and Technology [70]. Male cattleyak (Holstein Maiwa yak; n ¼ 3, named P1, P2, and P3) and male yak (Maiwa yak; n ¼ 3, named M1, M2, and M3) aged 12 months were sampled from a Maiwa yak population fed on a pasture in Hongyuan county, Sichuan province of China, in which cattleyak were F1 generations of Holstein and Maiwa yak. The epididymis of each animal was obtained by veterinary surgical operation and fat and connective tissues were removed. Epididymes were separated apart from testis by fine-scale dissection and were snap-frozen in liquid nitrogen (-196°C), transported to the laboratory and stored at -80°C until RNA isolation.

RNA Extraction, Library Preparation and Sequencing
Three biological replicates were used for yak and two biological replicates for cattleyak as in the previous studies [71,72]. Total RNA extraction was performed from each epididymis frozen samples of yak and cattleyak. RNA was extracted using Trizol (Life Technologies, LT) as per the manufacturer's protocol [73,74]. RNA degradation and DNA contamination was monitored on 1.5% agarose gel. The RNA concentration and purity were confirmed by using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) according to the manufacturer's instructions. The RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, USA).
A total amount of 1.5 µg RNA per sample was used as an input material for rRNA removal using the Ribo-Zero rRNA Removal Kit (Epicenter, Madison, WI, USA). Libraries sequences were generated using NEBNext R Ultra ™ Directional RNA Library Prep Kit for Illumina R (NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and Reverse Transcriptase. Second-strand cDNA synthesis was succeeded using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities after adenylating of 3' ends of DNA fragments. NEBNext Adapter with hairpin loop structures was ligated to prepare for hybridization. In order to insert fragments of preferentially 150-200 bp in length, the library fragments were purified with AMPure XP beads (Beckman Coulter, Beverly, USA). Then 3 µL of USER Enzyme (NEB, USA) was used with size-selected, adapter-ligated cDNA at 37°C for 15 min before PCR. PCR was then performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR.
An AcBot Cluster Generation System was used for the clustering of the index-coded samples by using TruSeq PE Cluster Kitv3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq Xten platform (Biomarker Technologies Co. LTD, Beijing, China) and pairedend reads were generated.

Data Processing, and Assembly of Yak and Cattleyak Transcriptome
Raw data (raw reads) of fast format were first processed through in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyzes were based on clean data with high quality. The specified reference genome used was cow UMD3.1, for the sequence alignment of the clean reads from yak and cattleyak and subsequent analysis. Cow (Bos taurus) UMD3.1 (GCA_000003055.3) reference genome was retrieved from ensemble database: http://www.ensembl.org/ Bos_taurus/Info/Index. The alignment of yak and cattleyak clean reads to reference genome UMD3.1 was performed using HISAT2 in an efficient alignment system. The transcriptome was assembled using the StringTie based on the reads mapped to the reference genome. StringTie (1.3.1) https://ccb.jhu.edu/software/stringtie/index.shtml was used to calculate FPKMs (fragments per kilobase of transcript per million fragments mapped), of transcripts abundance in each sample. The number of fragments extracted from one transcript is related to the amount of sequenced data, the length of the transcript, and the expression level of the transcript. In order for the number of fragments to truly reflect the expression level of the transcript, the number of fragments in the Mapped Reads Number and transcript length were normalized. StringTie uses Fragments per Kilobase of transcript per Million fragments mapped as a measure of transcript or gene expression level, and FPKM were calculated as follows: !"#$ = !"#$ !"#$%&'() !"##$% !"#$%&'() !"##"$%& ×!"#$%&"'(! !"#n!"ℎ(!") The gff compare program was used to compare the gene and transcripts with the annotation. Differential expression analysis of two groups of yak and cattleyak samples was performed using the DESeq R package (1.10.1). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value <0.01 and an absolute value of log2 (fold change) > 2 found by DESeq were assigned as differentially expressed. We finally chose Fold Change ≥ 2 and FDR < 0.05 as the screening criteria.

Functional Annotation and Functional Enrichment Analysis of Differentially Expressed Genes
For the functional annotation of the transcripts, the unigenes were blasted against the databases of Nr, Swiss-Prot, KEGG, and COG using BLAST program with an E-value ≤ 1e-5. Te Blast2GO program was used to annotate the major GO categories of genes with an E-value ≤ 1e-5 [75].

Pathway Enrichment Analysis
GO enrichment analysis Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented by the topGO R packages. For KEGG pathway enrichment analysis, we used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways [76].

qPCR Validation of mRNA-Seq
To validate the repeatability and reproducibility of gene expression data from RNA-sequencing, quantitative reverse transcriptase PCR (qRT-PCR) analysis of 10 DEGs was performed on the same samples. Based on their potential functional importance, 12 and 10 DEGs were selected for validation by qPCR from yak and cattleyak, respectively. The cDNA was prepared from the remaining total RNA after transcriptome sequencing according to the instructions of the SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). A SYBR Green assay was used to quantify the expression of each gene using SYBR Premix Ex Taq (TaKaRa, Dalian, China) in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Munich, Germany) and Actin as the internal control, with the following cycle conditions: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 20 s. Three yak and cattleyak biological replicates were analyzed for each gene with three technical replicates. Data were processed using the 2−ΔΔCt method [77] and analyzed statistically using the Student's ttest in Sigmaplot v.8 software to compare the difference between the yak and the cattleyak samples at P <0.05. The efficiency of aligning Reads with reference genome of each sample ranged from 88.77% to 91.24%.

CONCLUSION
In conclusion, we have shown that several families of genes were downregulated in the epididymides of infertile male cattleyak compared to yak. Many of these genes appear to be specifically involved in the regulation of spermatozoal maturation, motility, host defense and the regulation of ions in the epididymal lumen. This transcriptome dataset will enrich the transcriptomic information for male cattleyak, and provide fundamental support for future research on the molecular mechanisms governing male cattleyak infertility. In addition, a large number of DEGs were obtained, which can be useful for further functional analysis studies in cattleyak breeding research. Furthermore, our finding also revealed very important pathways that are suggested to be associated with infertility of male cattleyak. Especially, protein processing endoplasmic reticulum and oxidative phosphorylation are suggested to be further investigated for a better understanding of the molecular mechanism and etiology of infertility in male cattleyak.

HUMAN AND ANIMAL RIGHTS
Sample collection was carried out under licence in accordance with the Guidelines for Care and Use of Laboratory Animals of China.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data supporting the findings of the article is available in the Biomarker at www.biomarker.com.cn, with reference number BMK170616-F748-0101.

FUNDING
This work was funded by the National Natural Science Foundation of China (31601946) and Longshan Academic Talent Research Supporting Program of SWUST (17LZ X671 and 18lzx661).


text:
Field Application of Trichoderma Suspension to Control Cacao Pod Rot ( Phytophthora palmivora )

On cocoa, black pod rot disease is caused by a plant pathogen in the genus of Phytophthora (Guest, 2007; Vanegtern, Rogers, & Nelson, 2015). In Phytophthora genus, more than 80 species caused plant diseases, including P. palmivora, P. megakarya, P. citrophora, and P. capsici, are responsible for black pod rot on cacao (Vanegtern, Rogers, & Nelson, 2015). Cacao pod rot caused by fungal pathogen, Phytophthora palmivora, is an important disease in cacao plantations and contributes significant losses to global cocoa production (Guest, 2007; Vanegtern, Rogers, & Nelson, 2015). Phytophthora palmivora infects fruits from various stages of fruit development, starting from young fruit or fruit nipples to almost mature fruit (Muzuni, Indradewi, & Baharudin, 2015). From 20 to 30 % pod losses through black pod rot and ca. 10 % of trees annually through stem cankers are caused by P. palmivora (Guest, 2007). Biological control agents (Trichoderma) available in the nature are able to find their own targets, relatively cheap, safe for the environment and health and can be reproduced simply. Many Trichoderma species, commonly are able to produce spores and secondary metabolites like 6-pentylalpha-pyrone (6-PP), a lactone with antibiotic properties and coconut aroma (Hamrounı, Molınet, Dupuy, Masmoudı, & Roussos, 2017). Plants wield an arsenal of structurally diverse chemical compounds called secondary metabolites, the active secondary metabolite compounds were classified into several ARTICLE INFO

INTRODUCTION
On cocoa, black pod rot disease is caused by a plant pathogen in the genus of Phytophthora (Guest, 2007;Vanegtern, Rogers, & Nelson, 2015). In Phytophthora genus, more than 80 species caused plant diseases, including P. palmivora, P. megakarya, P. citrophora, and P. capsici, are responsible for black pod rot on cacao (Vanegtern, Rogers, & Nelson, 2015). Cacao pod rot caused by fungal pathogen, Phytophthora palmivora, is an important disease in cacao plantations and contributes significant losses to global cocoa production (Guest, 2007;Vanegtern, Rogers, & Nelson, 2015). Phytophthora palmivora infects fruits from various stages of fruit development, starting from young fruit or fruit nipples to almost mature fruit (Muzuni, Indradewi, & Baharudin, 2015). From 20 to 30 % pod losses through black pod rot and ca. 10 % of trees annually through stem cankers are caused by P. palmivora (Guest, 2007).
Biological control agents (Trichoderma) available in the nature are able to find their own targets, relatively cheap, safe for the environment and health and can be reproduced simply. Many Trichoderma species, commonly are able to produce spores and secondary metabolites like 6-pentylalpha-pyrone (6-PP), a lactone with antibiotic properties and coconut aroma (Hamrounı, Molınet, Dupuy, Masmoudı, & Roussos, 2017). Plants wield an arsenal of structurally diverse chemical compounds called secondary metabolites, the active secondary metabolite compounds were classified into several  (Ahmed et al., 2017). Fungi can be grown in liquid culture Broth Agar for various purposes such as for releasing metabolite extract, enzyme and antibiotics (Nevalainen, Kautto, & Te'o, 2014).
Related to the important role of Trichoderma to control plant disease, it is necessary to evaluate the use of Trichoderma suspension from T. harzianum and T. virens species to control cacao pod rot disease on cacao plantation.

MATERIALS AND METHODS
This research was carried out in the cacao farm in Pulo Hagu Village, Padang Tiji Subdistrict, Pidie District. Laboratory activities were conducted at the Plant Disease Laboratory, Faculty of Agriculture, Syiah Kuala University and the Organic Chemical Laboratory, Faculty of Math and Science, Syiah Kuala University. All activities were carried out from March to July 2017.
The culture isolates of T. harzianum and T. virens were isolates of the Laboratory of Plant Diseases, Faculty of Agriculture, Syiah Kuala University. Phytochemical extraction has been conducted in The Organic Chemical Laboratory, Faculty of Math and Science, Syiah Kuala University.
The field experiment design used a single pattern Randomized Block Design (RBD) consisting of three treatments, namely: without suspension (as control), suspensions of T. Harzianum and T. virens. Each treatment was replicated eight times and each replication consisting of three units of cacao plants. It was obtained 72 test units.

Trichoderma Suspension
Trichoderma harzianum and T. virens isolates that used in this study, as collection of the Plant Disease Laboratory, Faculty of Agriculture, Syiah Kuala University. The isolate was cultured aseptically by cutting a small portion of T. harzianum inoculums growth on PDA media using a scalpel knife and then transferred it to a Petridis containing PDA, incubated for five days at room temperature (25 °C).
For a week, Trichoderma was propagated on PDA media and then in liquid media. Liquid media consisted of 4 L of rice washing water, 1 L of coconut water, and 75 g of granulated sugar. All ingredients were boiled for 40-60 minutes, then put into sterile plastic bottle (which have been washed with 70 % alcohol), each plastic bottle filled with 2.5 L of liquid media, closed tightly and cooled by soaking in a bucket. After being cool, T. harzianum and T. virens as much as 10 ml were inoculated into each plastic bottle aseptically on laminar air flow. The plastic bottle was tightly closed to avoid contamination by microorganisms from outside. Liquid media were shaken by a shaker (Eyeglass) device for three weeks at 150 rpm. The shaking function was to produce metabolites and reduce the formation of mycelium. Trichoderma liquid formulation was used after three weeks.

Trichoderma Secondary Metabolite Application
The application of suspension of T. harzianum and T. virens was carried out after the last harvest by spraying suspension metabolites which had been diluted. Suspension applied to cacao plantations on the stems, branches, leaves, flowers and evenly on the rhizosphere using sprayer/solo. The application of secondary metabolites was applied in the morning (from 7:00 to 10:00 a.m.) with three times and 14 days of application interval. The dilution formula was described as formula 1.

Percentage of Affected Fruit
The attacked fruit that counted was cacao fruit infected by P. palmivora. The observation of the percentage of attacked fruit was carried out at 4, 6, 8, 10 and 12 WAA. The percentage of infected fruit was calculated based on the formula 2.

2)
Copyright © 2019 Universitas Brawijaya Where: P = percentage of fruit attacked; a = number of fruits attacked; b = number of whole fruits

Intensity of Affected Fruit
After calculating all fruits were classified to the affected fruit based on the percentage of the attack on each fruit (Fig. 1). The intensity of infected fruit was calculated based on the formula 3.

3)
Where: I = the intensity of the fruit attacked; ni = the number of pieces in a particular attack; si = the scale on certain attacks; N = the number of fruits observed; S = the highest scale value

Percentage of Reduction Area
The area of the initial spot was measured at 84 Days After Application (DAA) to determine the increase in the area of the spot in each treatment. The measurement repeated in the area of spotting at 87 DAA, by wrapping cacao fruit whose attack rates were 1-5 %, 6-20 % and > 20 % each one sample with plastic and given a broad marking of the initial spot, three days later a large area of spotting was added and marked. The plastic that has been marked was cut according to the size of the spot. Then the leaf area meter was inserted. The way to find out the area of the spot was the area of the spot on the 87 th gauge DAA reduced by the area of measurement to 84 DAA. To get the percentage reduction in speckle area was calculated by the formula 2 (Asrul, 2009):

4)
Where PPLB is Percentage of Reduction in Spots.

Data Analysis
Sigma Plot 12.0 versions was used to provide the graph and statistical analysis. The Analysis of variance was used to evaluate data. If there were found significances between each treatment, Duncan Multiple Range Test (DMRT) at 5 % of α is performed to check the significant differences between each treatment.

Phytochemical Test
The content of secondary metabolites in each species of Trichoderma spp. after the phytochemical test is shown in Table 1. Phytochemical test results show the presence of secondary metabolite content in Trichoderma spp. species (Table 1). However, the five species of Trichoderma spp. tested, there were no species containing terpenoids. This is indicated by the absence of red color when the five species were reacted with the Liebermann-Burchad reagent. Likewise Steroids, no fat-soluble complex molecules that are shown by four rings are joined together (Kumaresan, Karthi, Senthilkumar, Balakumar, & Stephen, 2015). While flavonoid compounds were also not found which marked by the absence of pink color after adding magnesium metal and a drop of hydrochloric acid in the final process of flavonoid tests.  However, alkaloid compounds were found in all Trichoderma species tested and was marked by the appearance of orange color after reacting with Dragendorff reagent, and the color was white after reacting with Mayer reagent. Alkaloids are secondary metabolite organic compounds found in nature. In fungi (especially endophytic fungi), alkaloids have properties such as antifungals and antiviral activity (Zhang et al., 2012).

Trichoderma Field Application Test Percentage of Affected Fruit
The average percentage of infected fruit due to the application of T. harzianum and T. virens suspension on observations 4, 6, 8, 10 and 12 WWA can be seen in the Table 2. The treatment without the application of Trichoderma suspension (T0) has the highest percentage of infected fruit in each observation ( Table 2). The highest percentage of infected fruit in T0 treatment was 56.04 % at 12 WAA and the lowest percentage of infected fruit in T1 treatment was 7.47 % at 12 WAA. The application of T. harzianum suspension could reduce the percentage of infected fruit at each observation by 15.17 % at 6 WAA, 27.29 % at 8 WAA, 41.98 % at 10 WAA and 48.57 % at 12 WAA while the suspension treatment of metabolite T virens reduced the percentage of infected fruit by 11.76 % at 6 MSA, 22.19 % at 8 WAA, 38.45 % at 10 WAA and 40.61 % at 12 WAA. The data of the percentage of infected fruit showed that the suspension treatment of T. harzianum was the best treatment in suppressing the percentage of infected fruit caused by P. palmivora. Biofungicides with active ingredients T. harzianum applied have the advantage of being able to stick to the soil and roots, protect the roots and are not dissolved by water, because Trichoderma produces different kinds of enzymes which play a major role in biocontrol activity like degradation of cell wall, tolerance to biotic or abiotic stresses, hyphal growth etc. (Waghunde, Shelake, & Sabalpara, 2016). It was also suspected that the metabolite suspension of T. harzianum and T. virens which was applied to the flower, leaves and roots of cacao could prevent pathogenic infection of P. palmivora from the beginning of flowering so that the fruit had been colonized with suspension of Trichoderma metabolites. Efficacy of foliar spray of T. harzianum and T. viride isolates against Fusarium graminearum causing head blight (head scab) of wheat (Panwar et al., 2014). Among the different species and isolates of Trichoderma evaluated worldwide for control of foliar diseases, one such as grey mold disease cause by Botrytis cinera (Sawant, 2014) and monilia infection on cocoa (Seng, Herrera, Vaughan, & McCoy, 2014).
The ability of Trichoderma to reduce the percentage of infected fruit is due to its ability to suppress pathogenic populations directly or indirectly. Directly through the mechanism of antibiotics, competition and mycoparasites, while indirectly through increasing plant resistance (Aneja, Gianfagna, & Hebbar, 2005;Benítez, Rincón, Limón, & Codón, 2004). Trichoderma sp. also has the ability to remove hydrolytic enzymes, a class of hydrolytic enzymes whose very important role in the process of mycoparasitism of some pathogenic fungi is chitinolytic enzymes, which consist of chitinase. Remarks: T0 = Without Trichoderma suspension, T1 = Suspension of T. harzanum, T2 = Suspension of T. virens, WAA = Week After Application, x̄ = Average, SD = Standard Deviation. The numbers followed by the same letters in the same column are not significantly different at the level of 5 % (DMRT test)

Intensity of Affected Fruit
The highest attacked fruit intensity was found in treatment K0 which was 51.44 % at 12 WAA and the lowest was in the treatment of T. harzianum suspension 5.40 % at 12 WAA (Table 3). Metabolite T. harzianum suspension application reduced the intensity of infected fruit by 6.18 % at 4 WAA, 16.73 % at 6 WAA, 25.11 % at 8 WAA, 40.34 % at 10 WAA and 46.04 % at 12 WAA while the suspension treatment of metabolite T. virens reduced the intensity of infected fruit by 5.08 % at 4 WAA, 13.65 % at 6 WAA, 19.88 % at 8 WAA, 37.16 % at 10 WAA and 38.02 % at 12 WAA, the data was obtained from the difference between treatment and control.
The concentration of T. virens 1 x 10 6 could reduce the severity of P. palmivora disease on cacao seedlings by 18 % and the concentration of T. virens 1x 10 8 decreased the severity of P. palmivora on the leaves of cacao seeds by 20 % (Sriwati et al., 2015). The application of formulations proved stable when diluted in water with 1 % and 0.5 % of sedimentation respectively after 24 hours showed an improved efficacy of Trichoderma spp. to control the cacao pods disease (Mbarga et al., 2012).
The effectiveness of Trichoderma in controlling cacao pod rot is due to the metabolites in the form of Alkaloid and other antibiotic produced by Trichoderma. Howell (2003) studied the molecular mechanism of lytic enzymes involved in T. harzianum activity and stated that T. harzianum could degrade fungal cell walls due to chitinase, gluconase and protease.  In addition T. harzianum can produce volatile (alkyl pyrones) compounds that are anti-fungal. While secondary metabolites produced by T. virens are pyrones, Isocyano Metabolites, Peptaibols, exoglyconase enzymes and endoglyconase, selobiase and chitinase (Vinale et al., 2014). Trichoderma virens is able to produce gliotoxins and gliovirin antibiotics that are specific in inhibiting oomycote (Howell & Puckhaber, 2005). Trichoderma metabolite have displayed beneficial effects to plants, increasing plant growth and development an inducing defense responses to abiotic stresses and pathogens (Cardoza et al., 2005). By seeing on metabolite compound group of T. harzianum and T. viren result (Table 1.), both contain only alkaloid compound, the point of mechanism role of antagonistic also working together with metabolite compound in ability to inhibited pathogen growth and development.

Percentage of Pod Spotting Area
The reduction percentage in spotting area between the metabolite suspension treatment of T. harzianum and the control was 47.24 % while the T. virens suspension treatment was 27.46 % (Fig. 2). This was confirmed with the research (Hanada, Pomella, Soberanis, Loguercio, & Pereira, 2009) states that the application Trichoderma spp. tended to decrease progressively on the surface of cacao pods, with a concomitant increase in the severity of pod disease. Sriwati et al. (2015) stated that T. harzianum had the highest level of colonizing cacao fruit compared to T. virens and T. asperellum.
Cacao plants that have been applied for suspension T. harzianum dan T. virens have been colonized by Trichoderma so that the area of the spot on the cacao fruit can be reduced. In addition Trichoderma is able to produce secondary metabolites in the form of viridine and trichomidine, and form of alkaloid both compounds capable of producing hydrolytic enzymes β1,3gluconase, chitinase and cellulase which can actively degrade other fungal cells; some of the mechanisms by which pathogenic microbes and various abiotic stresses as well as specific plant responses to pathogen attack, the genetic control of host-pathogen interactions (Mazid, Khan, & Mohammad, 2011). The biocontrol ability of Trichoderma is due to multiple factors, as they have the ability to produce a variety of extracellular lytic enzymes and many secondary metabolites (Cardoza et al., 2005).

CONCLUSION AND SUGGESTION
The application of T. harzianum suspension suppresses the percentage of infected fruit and the intensity of attacked fruit for 48.57 % and 46.04 % at 12 WAA respectively. In addition, based on the percentage reduction in the area of spot between control and T. harzianum suspension and T. virens are 47.24 % and 27.46 % at 87 WAA respectively. In addition, T. virens suppressed the percentage of infected fruit and the intensity of infected fruit for 40.61 % and 38.02 % at 12 WAA. Further research is needed on the effect of secondary metabolite combinations of various Trichoderma species in controlling cacao pod rot.

ACKNOWLEDGEMENT
This work was supported and funded by PSN, Penelitian Strategis Nasional Institusi, Ministry of Research, Technology and Higher Education No. 99/UN 11.2/PP/SP3/2017. Thanks to Yusmaini, Hagus Tarno and Yogo Setiawan for the laboratory assistant to conduct research, manuscript advices and manuscript writing process respectively.