2021 Vol. 36, No. 6
Display Method:
2021, 36(6): 619-628.
doi: 10.19303/j.issn.1008-0384.2021.06.001
Abstract:
Objective Basic characteristics and evolutional relationship on the phenylpropane metabolic pathway associated with the pepper blast disease that ill-affects the cultivation and production of the globally important spice, black peppers, were studied. Method The phenylpropane metabolic pathway was believed to be key to the blast resistance of black peppers, and phenylalanine ammonialyase (PAL) to be the crucial, rate-limiting enzyme in the pathway. Hence, based on the transcriptomics of a resistant black pepper germplasm the sequences of 14 PALs were identified and named PnPAL1-PnPAL14. Their bioinformatics and expression patterns were analyzed. Result The gene family had a theoretical isoelectric point between pH 5.76 and 9.77 with a molecular weight between 7.37441 kDa and 83.43107 kDa. Seven of the identified PnPALs contained 8 motifs and PLN02457 conserved domains. They belonged to the PnPAL family that consisted of many stress response elements, especially salicylic acid, methyl jasmonate, and other cis-acting elements, which are known to resist the pathogenic invasion. The PnPALs were located at the base of the phylogenetic tree, being relatively old and holding a status like black peppers. PnPAL10 expressed in an up-regulation trend after a pathogenic induction suggesting a close relation of the gene to the blast resistance of the plant. Conclusion A critical role the PnPAL family played in the blast resistance of black peppers was positively identified.
2021, 36(6): 629-635.
doi: 10.19303/j.issn.1008-0384.2021.06.002
Abstract:
Objective The coden usage bias and its infulence factor in the chloroplast genome of Liparis bootanensis were analyzed in this study. Method CodonW and CUSP software were used to analyze the codon usage bias based on 47 codon DNA sequences of the chloroplast genome of L. bootanensis. Result The GC content in the 3rd position of the codons was 27.99%, which was lower than those in the 1st and the 2nd. The effective number of codons ranged from 40.85 to 56.80, averaging 47.27. The ENC showed no significant correlation to GC1 and GC2 but a significant positive correlation to GC3. The correlation between GC12 and GC3 was not significant with a coefficient of 0.05. The frequency of ENC ratio of 47% genes was distributed between −0.05 and 0.05, while that of 53% genes outside -0.05 and 0.05 range. A PR2-plot analysis indicated the base usage frequencies to be A＞T and G＞C. There were 17 codons selected after screening. Conclusion The 3rd position of codons was rich in A or T. The codon usage bias was weak, complex, and affected by mutation and selection besides other factors.
2021, 36(6): 636-641.
doi: 10.19303/j.issn.1008-0384.2021.06.003
Abstract:
Objective To decipher the gene function, phytoalexin biosynthesis, and disease resistance mechanism of Dendrobium officinale (Do), the premnaspirodiene oxygenase Method RACE-PCR and RT-PCR were employed to clone the full-length cDNA and ORF of DoPO. ProtParam was used for the physiochemical determination, BLAST P on the amino acid homology, and MEGA 6.0 for the phylogenetic tree construction. The expressions of DoPO in different organs at flowering stage as well as in leaves at growth stages were examined by qPCR. Result The DoPO encoded 498 amino acids, 1 704 bp in length with an 1 497 bp ORF. The unstable protein had a molecular weight of 56.914 kDa belonging to the P450 superfamily. It was phylogenetically clustered in the same branch with Phalaenopsis equestris (XP 020571355) and Apostasia shenzhenica (PKA52400) and most closely related to P. equestris. The relative DoPO expression in the leaves was significantly higher than that in the stems, roots, or flowers at flowering stage (P＜0.01); and during a year, that in October significantly higher than that in either August or December (P＜0.01) and that in December significantly higher than in August (P＜0.05). Conclusion The full-length cDNA of DoPO was successfully cloned. Its relative expression in leaf peaked in October and was highest in leaves among the organs at flowering stage.
2021, 36(6): 642-650.
doi: 10.19303/j.issn.1008-0384.2021.06.004
Abstract:
Objective The optimized ISSR-PCR reaction system and appropriate polymorphic primers were established to facilitate the authentication, breeding, and genetic studies on Fritillaria hupehensis Hsiao et K.C. Hsia. Method A U12(43) uniform design method combined with a single factor screening test was employed to optimize the dosages of 2×Taq Master Mix, template DNA, and primer applied for ISSR-PCR and followed by a gradient temperature experiment to determine the primer annealing temperature. Result The optimal ISSR reaction system used 10.5 μL of 2×Taq Master Mix, 0.8 μL of template DNA (40.0 ng), 2.2 μL (2.5 μmol·L−1) of primers, and 6.5 uL of ddH2O for a 5 min sequencing at 94 ℃ and 40 cycles at 94 ℃ for 0.75 min, 54.3–60.0 ℃ for 0.75 min, and 72 ℃ for 1.5 min, followed by 10 min at 72 ℃ prior to storage at 4 ℃. Six stable polymorphic primers, UBC848, UBC850, UBC853, UBC857, UBC859, and UBC866 with the optimal annealing temperatures of 59.3, 58.2, 56.9, 54.3, 59.3, and 60.0 ℃, respectively, were selected. On 12 F. hupehensis germplasms from various locations, the optimized ISSR-PCR yielded stable and reliable results that showed abundant genetic diversity. Conclusion The optimized ISSR-PCR reaction system could be satisfactorily applied for the resource authentication and genetic analysis on F. hupehensis.
2021, 36(6): 651-657.
doi: 10.19303/j.issn.1008-0384.2021.06.005
Abstract:
Objective As an edible and medicinal fungus, Agaricus blazei Murrill is prone to accumulate heavy metals such as cadmium in production, resulting in excessive cadmium and other heavy metals. Breeding new varieties of Agaricus blazei Murrill with low cadmium and high quality is a scientific research target of great concern in the industry. Methods The J1 cultivar of A. blazei was exposed to 60Co radiation to obtain J37. The main agronomic traits, yield, and contents of amino acids, cadmium, lead, arsenic, and mercury in fruitarian bodies of J1 and J37 in the 1st to 4th tides were systematically compared. The effects of different growth algebras on fruitbody yield of Agaricus blazei Murrill J1 and J37 were also observed. Result (1) The flower weight, cover weight, cover thickness, cover diameter, cover height, stem weight, stem diameter and other agronomic traits of new strain J37 were significantly or extremely significantly higher than those of control (J1), while stem length was significantly lower than that of J1, and essential amino acid content was extremely significantly higher than that of J1; (2) Compared with J1, the yield of J37 in 1-6 generation (M1-6) was higher than that of J1, and the growth rate of fruite body yield in M1, M2, M3, M4, M5 and M6 generations of J37 was 30.11%, 33.58%, 39.46%, 39.45%, 34.14% and 33.53%, respectively, higher than that of J1. The difference reached extremely significant level; (3) Compared with J1, the contents of CD, Pb, Hg and AS in J37 fruite body decreased by 48.09%, 58.57%, 26.03% and 75.26%, respectively, and the differences were extremely significant. f A. blazei. The irradiation treatment appeared to have a potential for mushroom breeding. Conclusion The new agaricus blazei strain J37 showed good yield, high quality and safety.
2021, 36(6): 658-664.
doi: 10.19303/j.issn.1008-0384.2021.06.006
Abstract:
Objective Physiological responses to imposed simulated acid rain treatment were analyzed to determine the stress tolerance of two varieties of Taxus plants. Method Two-year-oldTaxus chinensis and T. yunnanensis, which are known to be resistant to acid rain, were treated by solutions of pH 4.5, 4.0, 3.5, and 3.0. Antioxidant enzyme activities and contents of chlorophyll (CHL), carotenoids (CAR), nutrients, malondialdehyde (MDA), and osmotic adjustment soluble proteins (SP) and sugars (SS) in leaves of the plants under treatments and control at pH 5.6 were measured. Result The activities of APX, PPO, SOD, POD, and CAT as well as the contents of SP and SS of the plants increased and followed by a decline, while MDA increased and CHL and CAR decreased as the treatment acidity increased. The stress tolerance of the two species differed, as T. chinensis peaked at pH 4.5 and T. yunnanensis at pH 4.0. However, neither of them could withstand pH 3.0 treatment without severe ill-effects physiologically. Conclusion In the simulated acid rain test, T. yunnanensis appeared to be more tolerant to the imposed stress than did T. chinensis.
2021, 36(6): 665-669.
doi: 10.19303/j.issn.1008-0384.2021.06.007
Abstract:
Objective A RT-PCR method for detecting new goose astrovirus (NGAstV) was developed. Method According to the ORF2 Result The optimized reaction system applied 10 μL premix, 1 μL each of ORF2 Conclusion The new RT-PCR method exhibited the specificity and repeatability that could satisfactorily be used for detecting and/or epidemiological studies relating to NGAstV.
2021, 36(6): 670-678.
doi: 10.19303/j.issn.1008-0384.2021.06.008
Abstract:
Objective Fermentation processes utilizing soymilk processing wastewater to prepare feed additives with a high viable bacteria count and desirable functionalities were tested for applications. Method Bacillus subtilis SB13 and B. licheniformis BL14 were cultured in the wastewater. Medium formulation and process optimization were conducted with a single factor experiment determined by the count of viable bacteria in the resulting preparations. Functions as an additive for chicken, pig, and cattle feeds of the preparations was evaluated by an in vitro simulated digestion and a DNS method. Result Both SB13 and BL14 grew normally in the soymilk wastewater. The optimized culture processes for SB13 and BL14 included sporulation temperatures at 35 ℃ for 72 h and 48 h, respectively. To facilitate dehydrating the fermentation broths, a protective agent of 10% skim milk powder was added in the medium for SB13 and 4% sucrose for BL14, as well as a carrier of 10% wheat bran for SB13 and 10% starch for BL14. After completion of the fermentation, the broths were freeze-dried and kept in sealed containers prior to functionality tests. The dried SB13 agent showed a viable bacteria count of 6.40×108 cfu·mL−1 and a survival rate at 97.71%, while BL14 a count of 9.80×108 cfu·mL−1 and a survival rate at 94.69%. Upon the artificial gastric juice and pancreatic juice treatments for 2 h, the two preparations had survived counts ranged from 2.55×107 to 13.10×107 cfu·mL−1. The endoglucanase activity of the preparations was more than 39% retained after the gastric juice treatment and 72% after the pancreatic juice treatment. Both agents displayed significant glycation on feed, and BL14 exhibited a significant inhibitory effect on Staphylococcus aureus. Conclusion Utilizing the wastewater from soymilk processing to culture B. subtilis SB13 and B. licheniformis BL14 resulted in preparations that could become a functional additive to forages for livestock and poultry industry. The process was cost effective and environmentally friendly.
2021, 36(6): 679-686.
doi: 10.19303/j.issn.1008-0384.2021.06.009
Abstract:
Objective Conserve and special functions of Rab4 protein of Colletotrichum graminicola (Cg) Method Based upon the principle of homologous recombination, CgRAB4 was knocked out from the DNA of Cg to obtain mutants with the target gene deleted. A hygromycin-resistance screening and PCR verification were performed on the mutants prior to a functional analysis on Cg development and pathogenesis. Result Three CgRAB4-deleted and one ectopic mutants were secured by the designed process. The phenotypic analysis revealed that the deletion promoted the hypha growth but did not affect the mycelial morphology, nor regulate the response to external stress or significant change in the spore production and morphology, appressorium germination or the pathogenicity on the mutants. Conclusion CgRAB4 was successfully removed from Cg to unveil the involvement of the gene in regulating the mycelial growth. Understanding on the roles of Rab proteins played in the development and pathogenesis of Cg provided a guiding direction for further research on the prevention and treatment of corn anthrax.
2021, 36(6): 687-691.
doi: 10.19303/j.issn.1008-0384.2021.06.010
Abstract:
Objective Melatonin contents in brown planthopper, Method HPLC-MS/MS was used to measure the melatonin contents in the 1st to the 5th instar nymphs as well as female and male macropterous and brachypterous adults of the insect. Result At nymph stage, N. lugens had the lowest average melatonin content of 378.5 pg·g−1 in the 1st and the 2nd instars, the highest of 856.6 pg·g−1 in the 3rd instar, 810.7 pg·g−1 in the 4th instar, and 574.5 pg·g−1 in the 5th instar. Adult N. lugens differed significantly on melatonin contents between male and female but not between the macropterous and brachypterous wing types. The male of macropterous and brachypterous wing types contained 978.3 pg·g−1 and 969.6 pg·g−1 melatonin, respectively, which were extremely significantly higher than the female counterparts at 76.1 pg·g−1 and 176.8 pg·g−1, respectively. Conclusion The melatonin contents in N. lugens as nymphs and adults were determined.
2021, 36(6): 692-698.
doi: 10.19303/j.issn.1008-0384.2021.06.011
Abstract:
Objective Effects of Chenopodium ambrosioides (L.) essential oil on the exoskeleton structure and the insecticide permeation of Plutella xylostella (Linn.) were studied to determine if the insecticidal efficacy on the moth could be improved by treatment with the essential oil. Method Changes on the exoskeleton ultrastructure of diamondback moth (DBM, P. xylostella) larvae upon treatment of the wormseed essential oil (WEO) or Azone (laurocapram) were examined under a scanning electron microscope. The permeabilities of subsequent separate application of 3 insecticides into the larvae by percutaneous absorption and penetration were determined by GC-MS. Result Using distilled water as control and acetone for comparison, WEO or Azone treatment produced significant alterations on the spines and the epicuticle waxy layer structure of DBM. The hair-like piles became sparse, the cuticle surface smoothed, and the lower part of spines folded or cracked. Prolonged treatment significantly increased the penetration of the 3 subsequently applied insecticides through DBM exoskeleton. In 24h, the transdermal absorption rate of fipronil had reached 99.34%, which was significantly higher than control（55.12%）. Conclusion The enhancing effect of WEO pretreatment on the transdermal penetration of subsequently applied insecticide on DBM was similar to that of Azone. The enhancement on fipronil penetration was also found on trichlorfon and chlorpyrifos permeation. Similar results were, therefore, expected for the cases involving other organophosphorus, phenylpyrazole, and thiophosphate insecticides.
2021, 36(6): 699-705.
doi: 10.19303/j.issn.1008-0384.2021.06.012
Abstract:
Objective Effect of long-term application of pig manure biogas slurry on the abundance and seasonal variations of antibiotic resistance genes (ARGs) presented in the soil was studied. Method Soil specimens at the fields using biogas slurry of pig manure for fertilization for many years were collected in four seasons to compare with those at a nearby location without such practice. The presence and abundance of tetracycline and sulfonamide ARGs in the soils were determined by quantitative PCR. Result There were 11 to 13 classes of ARGs detected in the biogas slurry fertilized soils. The ARG diversity was significantly greater in them than control with a relative abundance of tetG, sul1 and sul2 genes. The increased levels of tetracycline and sulfonamide ARGs also showed significant seasonal variations with the highest relative abundance in spring. Long application of biogas slurry on land resulted in abundant tetG, tetZ, tetM, tetO, tetW, sul1, and sul2 genes, which were the dominant ARGs year-round, in the soil. Conclusion Long-term biogas slurry application could elevate the ARG level in the soil. Therefore, it was highly recommended the waste material utilization be optimized by a fermentation process prior to fertilization for antibiotic contamination mitigation on farmland.
2021, 36(6): 706-712.
doi: 10.19303/j.issn.1008-0384.2021.06.013
Abstract:
Objective Culture methods for and detection of the antibiotic resistance genes (ARGs) in the endophytic bacteria of Azolla were established to monitor and find means to curtail the drug pollution in aquatic environment. Method Three selected media were used to culture the endophytic bacteria in Azolla microphylla for metagenome sequencing and bioinformatical analysis on ARGs. Result A total of 48.66G clean data was obtained to yield 54 180 predicted ORFs. Through the taxonomic information database NR, 1 712 microbial species were annotated belonging to 38 phyla. On species abundance of the bacteria from the 3 cultures, Proteobacteria accounted for more than 99.14% of all at the phyla level, while at genus level, Herbaspirillum approximately 91% and Burkholderia 1.2%. Blasted with the ARDB database, 3 samples were annotated with 10 genes associated with the resistance to bacitracin and chloramphenicol. Compared to CARD, there were 212 antibiotic related genes (AROs) found in the samples. The genes resistant to antibiotics (AR) accounted for more than 88% of total AROs, while those sensitive to antibiotics (AS) approximately 7% and those resistant to target antibiotics (AT) 4-6%. Conclusion The community structure and ARG types of the endophytic bacteria in Azolla cultured in the 3 different media were basically same.
2021, 36(6): 713-718.
doi: 10.19303/j.issn.1008-0384.2021.06.014
Abstract:
Objective Auxin IAA-induced sporulation of Azolla was investigated. Method Under natural conditions in a netted chamber, auxin IAA solutions in concentration of 0 (control), 1, 5, 10, 15, or 20 μg·mL−1 was sprayed once-a-week on Azolla filiculoides Lamarck 1001, A. caroliniana wild 3001, and A. microphylla Kaulfus 4018 during vegetative growth period for the duration until sporocarps appeared . Result After five months of spraying with 1, 5, or 10 μg·mL−1 IAA solution, sporocarps began to form only on Azolla 1001. The sporulation rate, spore count, and female/male ratio were 15.0%, 6.31, and 0.62 ∶ 1, respectively, under the 5 μg·mL−1 application, which significantly differed from 6.3%, 4.12, and 0.53 ∶ 1, respectively, under the 10 μg·mL−1 application, 4.7%, 3.90, and 0.51 ∶ 1, respectively, under the 1 μg·mL−1 application, and control. In addition, the size and C/N ratio of Azolla 1001 significantly increased by the spraying, as compared to control, whereas none observed on the other two ferns. Conclusion Auxin IAA could induce the sporulation of A. filiculoides Lamarck 1001. Spraying IAA at the concentration of 5 μg·mL−1 on the ferns appeared feasible for field application.
2021, 36(6): 719-727.
doi: 10.19303/j.issn.1008-0384.2021.06.015
Abstract:
Objective Decomposition and nutrient release of green manure after returning to soil in citrus field were studied. Method Using nylon mesh bags to contain Dolichos lablab, Astragalus sinicus, Viciavillosa Rothvar, Medicago sativa, or Lolium multiflorum and plowed into the soil at a depth of 20 cm. Soil samples were taken on the 20th , 40th, 60th, 80th, and 100th day after manure turning over to determine carbon, nitrogen, phosphorus, and potassium contents. Carbon/nitrogen ratio, material degradation, and nutrient release were tabulated for analysis. Result The manure decomposition and nutrient release were at a high rate in 0–20 d after the deposit but slowed down from the 20th to the 100th day. The rate of material decay reached 35.52%–73.96% after 20 d, and 79.13%–90.25% after 100 d. For the 5 green manures, the releases of C, N, P, and K ranged 38.10%–78.59%, 66.92%–87.63%, 28.60%–80.92%, and 80.22%–96.72%, respectively, in 20 d, and 80.90%–95.48%, 94.63%–97.64, 59.66%–96.28%, and 94.46%–99.64%, respectively, in 100 d. The cumulative nutrient release was K＞N＞C＞P, and the C/N ratio increased as the decomposition progressed. Conclusion Among the 5 green manures, A. sinicus displayed the highest cumulative decay rate, D. lablab the most cumulative N and K releases, and L. multiflorum the greatest cumulative C and P releases.
2021, 36(6): 728-734.
doi: 10.19303/j.issn.1008-0384.2021.06.016
Abstract:
Objective In order to increase the ergothioneine content of Pleurotus eryngii 528 fermented products, the nutritional formula of Pleurotus eryngii 528 was screened to provide a theoretical basis for the development of high content of ergothioneine in submerged fermentation of Pleurotus eryngii . Method The suitable carbon and nitrogen sources for Pleurotus eryngii 528 mycelium growth were discussed based on the solid culture mycelium growth rate and mycelium morphology. A single factor test was conducted to study the nutritional formula of mycelium growth in Pleurotus eryngii 528 submerged fermentation, with the indexes of the dry weight of the fermented product, the sedimentation and the initial pH value of the medium. The effects of different amino acids on ergothioneine content in submerged fermentation broth were investigated. Finally, L9(34) orthogonal test was used to analyze the formula of submerged fermentation medium for increasing ergothioneine content in Pleurotus eryngii 528. Result Corn grits and peptone were the suitable carbon and nitrogen sources for the growth of Pleurotus eryngii 528. In the liquid fermentation of Pleurotus eryngii 528, the dry weight of the fermentation product in the liquid culture medium of corn grits powder 2.0% and peptone 2.0% reached 33.30±0.81 g·L−1 , which was significantly higher than that of other carbon and nitrogen source combinations, and the sediment condition and initial pH value were more suitable for Pleurotus eryngii 528 liquid culture. Aspartic acid, cysteine, histidine, arginine and glutamic acid improved significantly the ergothioneine yield of Pleurotus eryngii 528 fermented product. The content of ergothioneine in the liquid fermentation of Pleurotus eryngii induced by histidine was the highest, which was 3.73 times of the control group, and was significantly higher than other amino acids. Conclusion The optimum medium formula of ergothioneine biosynthesis by submerged fermentation of Pleurotus eryngii 528 was as follows: corn grits 2.0%, peptone 1.5%, histidine 0.15%, wheat bran powder 1.0%, KH2PO4 0.1%, MgSO4 0.1%, glutamic acid 0.1% and vitamin B6 0.1%. Under these conditions, the content of ergothioneine in Pleurotus eryngii fermentation reached 20.50±1.80 mg·L−1.
