Phenotypic characterization and genetic mapping of the dwarf mutant m34 in maize

Plant height is one of the most important agronomic traits associated with yield in maize.In this study,a gibberellins(GA)-insensitive dwarf mutant,m34,was screened from inbred line Ye478 by treatment with the chemical mutagen ethylmethanesulfonate(EMS).Compared to Ye478,m34 showed a dwarf phenotype with shorter internodes,and smaller leaf length and width,but with similar leaf number.Furthermore,m34 exhibited smaller guard cells in internodes than Ye478,suggesting that smaller cells might contribute to its dwarf phenotype.Genetic analysis indicated that the m34 dwarf phenotype was controlled by a recessive nuclear gene.An F2 population derived from a cross between m34 and B73 was used for mutational gene cloning and this gene was mapped to a chromosome region between umc2189 and umc1553 in chromosome 1 bin1.10,which harbored a previously identified dwarf gene Zm VP8.Sequencing analysis showed a nucleotide substitution(G1606 to A1606)in the sixth exon of ZmVP8,which resulted in an amino acid change(E531 to K531)from Ye478 to m34.This amino acid change resulted in anα-helix changing to aβ-sheet in the secondary protein structure and the‘SPEC’domain changed to a‘BOT1NT’domain in the tertiary protein structure.Taken together,these results suggested that m34 is a novel allelic mutant originally derived from Ye478 that is useful for further ZmVP8 functional analysis in maize.     

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.     

The synergistic advantage of combining chloropicrin or dazomet with fosthiazate nematicide to control root-knot nematode in cucumber production

The highly-damaging root-knot nematode(Meloidogyne spp., RKN) cannot be reliably controlled using only a nematicide such as fosthiazate because of increasing pest resistance. In laboratory and greenhouse trials, we showed that chloropicrin(CP) or dazomet(DZ) synergized the efficacy of fosthiazate against RKN. The combination significantly extended the degradation half-life of fosthiazate by an average of about 1.25 times. CP or DZ with fosthiazate reduced the time for fosthiazate to penetrate the RKN cuticle compared to fosthiazate alone. CP or DZ combined with low or medium rate of fosthiazate increased the total cucumber yield, compared to the use of each product alone. A low-dose fosthiazate with DZ improved total yield more than a low dose fosthiazate with CP. Extending the half-life of fosthiazate and reducing the time for fosthiazate or fumigant to penetrate the RKN cuticle were the two features that gave the fumigant-fosthiazate combination its synergistic advantage over these products used singularly. This synergy provides the opportunity for farmers to use a low dose of fosthiazate which lowers the risk of RKN resistance. Farmers could combine DZ at 30 g m~(-2) with fosthiazate at a low rate of 0.375 g m~(-2) to control RKN and adequately control two major soil-borne diseases in cucumber greenhouses.     

Estimates on nitrogen uptake in the subsequent wheat by above-ground and root residue and rhizodeposition of using peanut labeled with 15N isotope on the North China Plain

Leguminous crops play a vital role in enhancing crop yield and improving soil fertility. Therefore, it can be used as an organic N source for improving soil fertility. The purpose of this study was to (i) quantify the amounts of N derived from rhizodeposition, root and above-ground biomass of peanut residue in comparison with wheat and (ii) estimate the effect of the residual N on the wheat-growing season in the subsequent year. The plants of peanut and wheat were stem fed with ¹⁵N urea using the cotton-wick method at the Wuqiao Station of China Agricultural University in 2014. The experiment consisted of four residue-returning strategies in a randomized complete-block design: (i) no return of crop residue (CR0); (ii) return of above-ground biomass of peanut crop (CR1); (iii) return of peanut root biomass (CR2); and (iv) return of all residue of the whole peanut plant (CR3). The 31.5 and 21% of the labeled ¹⁵N isotope were accumulated in the above-ground tissues (leaves and stems) of peanuts and wheat, respectively. N rhizodeposition of peanuts and wheat accounted for 14.91 and 3.61% of the BG¹⁵N, respectively. The ¹⁵N from the below-ground ¹⁵N-labeled of peanuts were supplied 11.3, 5.9, 13.5, and 6.1% of in the CR0, CR1, CR2, and CR3 treatments, respectively. Peanut straw contributes a significant proportion of N to the soil through the decomposition of plant residues and N rhizodeposition. With the current production level on the NCP, it is estimated that peanut straw can potentially replace 104 500 tons of synthetic N fertilizer per year. The inclusion of peanut in rotation with cereal can significantly reduce the use of N fertilizer and enhance the system sustainability.     

Species-specific COI primers for rapid identification of a globally significant invasive pest,the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.     

Sublethal effects of sulfoxaflor on the fitness of two species of wheat aphids,Sitobion avenae(F.) and Rhopalosiphum padi(L.)

Sitobion avenae(F.) and Rhopalosiphum padi(L.) are two important pests of wheat in China. They typically coexist in fields during the late period of wheat growth. Sulfoxaflor is a novel sulfoximine insecticide that demonstrates broad-spectrum efficacy, especially in targeting sap-feeding insects. This study was carried out to investigate the sublethal effects of sulfoxaflor on the development, longevity, and reproduction of two species of wheat aphids. Our results showed that sublethal concentrations of sulfoxaflor did not cause significant effects on the fecundity or the longevity of the parent generation(F_0 generation) of either S. avenae or R. padi. However, it caused transgenerational sublethal effects. For S. avenae, adult longevity of F_1 generation was significantly decreased. No significant differences were observed on the population parameters of S. avenae in the F_1 generation. For R. padi, the adult preoviposition period(APOP) and the total preoviposition period(TPOP) of F_1 generation were significantly reduced. The mean generation time(T) was significantly reduced in the R. padi F_1 generation. What's more, the intrinsic rate of increase(r_m) and the finite rate of increase(λ) were significantly increased in the R. padi F_1 generation. Taken together, these results suggest that exposure to the LC_(25) of sulfoxafl had no effects on the parent generation of S. avenae or R. padi, but it reduced adult longevity of S. avenae as a negative effect and increased the r_m and λ of R. padi in the first progeny generation, which may have an impact on the population dynamics of R. padi.     

Mitochondrial DNA diversity and origin of indigenous pigs in South China and their contribution to western modern pig breeds

Indigenous pigs in South China are valuable genetic resources with many specific and unique characters, which have played an important role in the establishment of some western modern pig breeds. However, the origin and genetic diversity of indigenous pigs in South China have not been fully understood. In the present study, we sequenced 534 novel mitochondrial DNA(mt DNA) D-loop and assembled 54 complete mitogenome sequences for all 17 indigenous pig breeds from Fujian, Guangdong, Guangxi and Hainan in South China. These data were analyzed together with previously published homologous sequences relevant to this study. We found that all 13 coding genes of the mitogenomes were under purifying selection, but ND1 had the most variable sites and CYTB contained the most non-synonymous SNPs. Phylogenetic analysis showed that all indigenous pigs in South China were clustered into the D haplogroup with D1 a1, D1 b, D1 c and D1 e sub-haplogroups found to be dominant. Haplotype and nucleotide diversities of D-loop sequences ranged from 0.427 to 0.899 and from 0.00342 to 0.00695, respectively, among which all pigs in Guangdong had the lowest diversity. The estimates of pairwise FST, gene flow(Nm) and genetic distance(Da) indicated that most of these indigenous pig breeds differentiated from each other significantly(P0.05). Among the western modern breeds, Berkshire and Yorkshire had significant Asian matrilineal footprints from indigenous pigs in South China, especially the Spotted pigs distributed in Guangdong and Guangxi. The neutrality test(Fu's F_S) indicated that indigenous pigs from Fujian and Guangxi had gone through recent population expansion events(P0.05). It is concluded that indigenous pigs in South China were most likely derived from the Mekong region and the middle and downstream regions of Yangtze River through Guangxi and Fujian. Our findings provide a complete and in-depth insight on the origin and distribution pattern of maternal genetic diversity of indigenous pigs in South China.     

GsMAPK4, a positive regulator of soybean tolerance to salinity stress

Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clarified. In this study, a proteomic technology based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase (GsGT4), 4-coumarate, coenzyme A ligase (Gs4CL1), mitogen-activated protein kinase 4 (GsMAPK4), dehydration responsive element binding protein (GsDREB1), and soybean cold-regulated gene (GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4 (GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which confirmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.     

Effect of exogenous GA_3 on flowering quality,endogenous hormones,and hormone-and flowering-associated gene expression in forcing-cultured tree peony(Paeonia suffruticosa)

Gibberellins(GAs) promote flowering in the forcing-cultured tree peony(Paeonia suffruticosa), however, the mechanism of regulating flowering is not fully understood. In this study, exogenous GA_3 was applied to five-year-old Luoyang Hong plants to explore responses in terms of endogenous hormones, flowering quality, and the hormone-and flowering-associated gene expression. Exogenous GA_3 application significantly promoted flower bud development and new branch growth, as well as improved flowering quality. Exogenous GA_3 application also stimulated the synthesis of endogenous GA_3 and indole-3-acetic acid(IAA) but reduced abscisic acid(ABA) levels. To further elucidate the regulatory mechanism, eight genes for GA biosynthesis and signaling, including Ps CPS, Ps KS, Ps GA_3 ox, Ps GA2 ox, Ps GID1 b, Ps GID1 c, Ps DELLA, and Ps GID2 were cloned for the first time, and sequence analysis was also performed. The results suggested that all the cloned genes have conserved structure as each homologous gene reported in the other species. Phylogenetic trees constructed by the each cloned gene showed that the phylogenetic evolutionary relationship of P. suffruticosa was closely related to Vitis vinifera. The expression patterns of the above genes, and genes for ABA and IAA biosynthetic and signaling, and the flowering time were also investigated. Most of the above genes showed higher expression in the control buds than those in the GA_3 treated buds at six developmental stages, whereas the expression levels of PsSOC1 and PsSPL9 were up-regulated by GA_3 treatment. The results also showed that the GA-biosynthetic and signaling pathways are conserved in tree peony, and the PsCPS, PsGA_3 ox, PsGA2 ox, PsGID1, PsDELLA, and PsGID2 genes are necessary for feedback regulation of GAs. Furthermore, hormone changes promoted PsSOC1 and PsSPL9 expression, and repressed PsSVP expression, which contributed to the improvement flowering quality in tree peony of forcing culture.     

Evaluating effective Trichoderma isolates for biocontrol of Rhizoctonia solani causing root rot of Vigna unguiculata

The highly diverse genus Trichoderma has provided many formulations that are alternatives to the chemical pesticides in agriculture. The present study was undertaken to investigate the biocontrol potential of eight Trichoderma species, T. atrobrunneum, T. guizhouense, T. paratroviride, T. pyramidale, T. rufobrunneum, T. simmonsii, T. thermophilum and T. viridulum, against the phytopathogenic fungus Rhizoctonia solani. Trichoderma isolates were first evaluated in vitro by dual culture tests for their antagonism, mycoparasitic ability and antifungal activity against R. solani. Their growth promoting potential was further assessed in relation to phosphate solubilization, indole acetic acid and siderophore production. Five of the isolates were selected and evaluated for their abilities to prompt plant growth and to control R. solani infecting Vigna unguiculata(cowpea) seedlings in vivo. Two most effective isolates, T. guizhouense 9185 and T. simmonsii 8702, significantly(P0.05) reduced the disease severity incidences(36.6 and 45.0%, respectively) and promoted plant growth, which have good prospects for application.     

Characterization of TaCOMT genes associated with stem lignin content in common wheat and development of a gene-specific marker

Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA(gDNA) sequences of TaCOMT genes located on chromosomes 3 A, 3 B, and 3 D were cloned by homologous cloning. Two allelic variants, TaCOMT-3 Ba and TaCOMT-3 Bb, were identified and differed by a 222-bp insertion/deletion(InDel) in the 3′-untranslated region(3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as Ta COMT-3 BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3 Ba was significantly(P0.01) higher than that of those with TaCOMT-3 Bb, and the marker TaCOMT-3 BM could be effectively used in wheat breeding.     

A rapid,simple, and sensitive immunoagglutination assay with silica nanoparticles for serotype identification of Pseudomonas aeruginosa

An agglutination test based on colored silica nanoparticles (colored SiNps) was established to detect serotypes of Pseudomonas aeruginosa. Monodisperse colored SiNps were used as agglutination test carriers. The colored SiNps were prepared through reverse microemulsion with reactive dyes, sensitized with 11 kinds of mono-specific antibodies against P. aeruginosa, and denoted as IgG-colored SiNps. Eleven kinds of IgG-colored SiNps were individually mixed with P. aeruginosa on a glass slide. Different serotypes of P. aeruginosa could be identified by agglutination test with evident agglutination. The P. aeruginosa could be detected in a range from 3.6 × 10⁵ to 3.6 × 10¹² cfu mL^?1. This new agglutination test was confirmed to be a specific, sensitive, fast, easy-to-perform, and cost-efficient tool for the routine diagnosis of P. aeruginosa.     

Residue management induced changes in soil organic carbon and total nitrogen under different tillage practices in the North China Plain

Crop residue retention has been considered a practicable strategy to improve soil organic carbon(SOC) and total nitrogen(TN), but the effectiveness of residue retention might be different under varied tillage practices. To evaluate the effects of residue management on the distribution and stocks of SOC and TN under different tillage practices, a bifactorial experiment with three levels for tillage practices(no-tillage, rotary tillage, and conventional tillage) and two levels for residue managements(residue retention and residue removal) was conducted in the North China Plain(NCP). Results showed that after a short experimental duration(3–4 years), concentrations of SOC and TN in the 0–10 cm layer were higher under no-tillage than under conventional tillage, no matter whether crop residues were retained or not. Residue retention increased SOC and TN concentrations in the upper layers of soil to some degree for all tillage practices, as compared with residue removal, with the greatest increment of SOC concentration occurred in the 0–10 cm layer under rotary tillage, but in the 10–30 cm layer under conventional tillage. The stocks of SOC in the 0–50 cm depth increased from 49.89 Mg ha–1 with residue removal to 53.03 Mg ha–1 with residue retention. However, no-tillage did not increase SOC stock to a depth of 50 cm relative to conventional tillage, and increased only by 5.35% as compared with rotary tillage. Thus, residue retention may contribute more towards SOC sequestration than no-tillage. Furthermore, the combination between residue retention and no-tillage has the greatest advantage in enhancing SOC and TN in the NCP region.     

Effects of the severity and timing of basal leaf removal on the amino acids profiles of Sauvignon Blanc grapes and wines

The effects of the severity and timing of leaf removal(LR) on the amino acids of Sauvignon Blanc grapes and wines were studied during the 2017 growing season. High-performance liquid chromatography(HPLC) was used to analyze the amino acids profiles of grape berries and wines. The basal leaves were removed at three time points(40, 56 and 72 days after flowering, named LR40, LR56 and LR72, respectively) at two severity levels(one at which the first, third, and fifth basal leaves of each shoot were removed(50% level); and another at which the first six basal leaves were removed(100% level)). The results showed that leaf removal had little impact on total soluble solids(°Brix), titratable acidity, pH or berry weight. The LR72-50% treated grapes had higher berry weight, titratable acidity and °Brix than those of the other treatments. The highest concentrations of total amino acids and of total amino acids except proline were detected in LR72-50% treated grapes(2 952.58 and 2 764.36 mg L~(-1), respectively); the lowest were detected in LR72-100% treated grapes(2 172.82 and 2 038.71 mg L~(-1), respectively). LR72-50% treatment significantly promoted the synthesis of aspartic acid, serine, arginine, alanine, aminobutyric acid and proline at both severity levels for grapes, the concentrations of all of these amino acids were increased relative to the control concentrations. The LR72-50%, LR40-100% and LR72-100% treated wines had higher total amino acids concentrations and higher concentrations of some individual amino acids, such as arginine, alanine and serine, than did the control wines. Of all the amino acids studied, glycine, tyrosine, cysteine, methionine and lysine were not significantly influenced by the timing or severity basal defoliation in grapes and wines. The present study reveals the effects of the timing and severity of leaf removal on the amino acids profiles of grapes and wines.     

Soil macroaggregates and organic-matter content regulate microbial communities and enzymatic activity in a Chinese Mollisol

The formation and turnover of macroaggregates are critical processes influencing the dynamics and stabilization of soil organic carbon (SOC). Soil aggregate size distribution is directly related to the makeup and activity of microbial communities. We incubated soils managed for >30 years as restored grassland (GL), farmland (FL) and bare fallow (BF) for 60 days using both intact and reduced aggregate size distributions (intact aggregate distribution (IAD)<6 mm; reduced aggregate distribution (RAD)<1 mm), in treatments with added glucose, alanine or inorganic N, to reveal activity and microbial community structure as a function of aggregate size and makeup. Over a 60-day incubation period, the highest phospholipid fatty acid (PLFA) abundance was on day 7 for bacteria and fungi, on day 15 for actinomycete. The majority of the variation in enzymatic activities was likely related to PLFA abundance. GL had higher microbial abundance and enzyme activity. Mechanically reducing macroaggregates (>0.25 mm) by 34.7% in GL soil with no substrate additions increased the abundance of PLFAs (average increase of 15.7%) and activities of β-glucosidase (increase of 17.4%) and N-acetyl-β-glucosaminidase (increase of 7.6%). The addition of C substrates increased PLFA abundance in FL and BF by averages of 18.8 and 33.4%, respectively, but not in GL soil. The results show that the effect of habitat destruction on microorganisms depends on the soil aggregates, due to a release of bioavailable C, and the addition of substrates for soils with limited nutrient availability. The protection of SOC is promoted by larger size soil aggregate structures that are important to different aggregate size classes in affecting soil C stabilization and microbial community structure and activity.     

Application of virus-induced gene silencing for identification of FHB resistant genes

Virus-induced gene silencing(VIGS) showed several advantages to identify gene functions such as short experimental cycle, more broad hosts, etc. In this study, the feasibility and efficiency of employing Barley stripe mosaic virus(BSMV)-based VIGS system to evaluate Fusarium head blight(FHB) resistance were explored in wheat. With variable conditions tested, it showed that the maximal silencing efficiency 78% on spike was obtained when the recombinant BSMV was inoculated on flag leaf at flagging stage. However, the plant may reduce its own immunity to FHB when inoculated with BSMV. To induce this impact, different Fusarium graminearum strains were tested and SF06-1 strain was selected for FHB resistance evaluation. Using this system, Ta AOC, Ta AOS, and Ta OPR3 involved in jasmonic acid(JA) signaling pathway were identified to positively regulate FHB resistance, which was underpinned by the results when silencing Ta AOS in wheat by stable transgenic plants.     

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

Laccase(EC 1.10.3.2)is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2(PxLac2),from the third instar larvae using polymerase chain reaction(PCR)and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR(qPCR).The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant(K_m)of 0.97 mmol L~(-1),maximal reaction speed(V_m)of 56.82 U mL~(-1),and activation energy(E_a)of 17.36 kJ mol~(-1) to oxidize2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt).Type II copper enhanced laccase activity below 0.8mmol L~(-1) and reduced enzyme activity above 0.8 mmol L~(-1) with an IC_(50) concentration of 1.26 mmol L~(-1).This study provides insights into the biological function of laccase.