Acquisition and Transmission of Fusarium oxysporum f. sp. vasinfectum VCG 0114 (Race 4) by Stink Bugs.

Fusarium oxysporum f. sp. vasinfectum VCG 0114 (race 4; i.e., FOV4) is an emerging pathogen that causes severe root rot and wilt of cotton. FOV4 is seed-borne, but the mode of seed invasion is uncertain. In an initial study, seeds in bolls that were puncture inoculated with FOV4 conidia when they were 25- or 30-days old became infected but remained viable. Because stink bugs can ingest and introduce bacterial and yeast pathogens into cotton bolls, we hypothesized that stink bugs may ingest and transmit FOV4. Southern green stink bugs and brown stink bugs were exposed to potato dextrose agar cultures of FOV4 and subsequently caged with cotton bolls to assess transmission potential. Both species fed on the cultures and acquired FOV4, and brown stink bugs transmitted FOV4 to cotton bolls. Thus, management of FOV4 may require management of stink bugs to mitigate the spread of the disease in cotton.

Genetic Diversity and Pathogenicity of Verticillium dahliae Isolates and Their Co-occurrence with Fusarium oxysporum f. sp. vasinfectum Causing Cotton Wilt in Xinjiang, China.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from Verticillium dahliae. Eighty-five V. dahliae isolates were obtained from wilted cotton plants collected from eight counties in Xinjiang. The isolates were assessed for genotypic diversity by DNA sequence analysis and PCR molecular genotyping with specific markers for race 1, race 2, defoliating (D) pathotype, nondefoliating (ND) pathotype, and mating type idiomorph Mat1-2. Isolates belonged to lineages 1A or 2B, with three subgenotypes found in each lineage. All isolates tested positive for race 2 and Mat1-2 markers. All isolates in lineage 2B tested positive for the ND pathotype marker but only isolates in the major subgenotype in lineage 1A tested positive for the D pathotype marker. Pathogenicity assays on Gossypium hirsutum 'Acala 44' demonstrated no significant difference among subgenotypes within each lineage. Isolates in lineage 1A caused greater shoot weight reductions, percent leaf drop, and percent diseased leaves than isolates in lineage 2B. One isolate in each lineage for 1A and 2B was avirulent. Isolates in lineage 1A caused greater than 50% leaf drop and a 17-g shoot weight reduction compared with a 9% leaf drop and a 6-g shoot weight reduction by isolates in lineage 2B. Overall, 42% of the V. dahliae isolates from Xinjiang were D pathotype but the percentage varied widely among locations. Two plants had both pathotypes. Nineteen isolates of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) also were recovered from wilted plants in Xinjiang. Two plants had both Verticillium wilt and Fusarium wilt pathogens. Both pathogens should be considered when using or developing wilt resistant or tolerant materials for Xinjiang.

Detection and Characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (Race 4) Isolates of Diverse Geographic Origins.

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense 'Pima S-7'. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar 'Stoneville 474' or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid.

Fusaric acid (FA) produced by Fusarium oxysporum plays an important role in disease development in plants, including cotton. This non-specific toxin also has antibiotic effects on microorganisms. Thus, one expects a potential pool of diverse detoxification mechanisms of FA in nature. Bacteria and fungi from soils infested with Fusarium and from laboratory sources were evaluated for their ability to grow in the presence of FA and to alter the structure of FA into less toxic compounds. None of the bacterial strains were able to chemically modify FA. Highly FA-resistant strains were found only in Gram-negative bacteria, mainly in the genus of Pseudomonas. The FA resistance of the Gram-negative bacteria was positively correlated with the number of predicted genes for FA efflux pumps present in the genome. Phylogenetic analysis of predicted FA resistance proteins (FUSC, an inner membrane transporter component of the efflux pump) revealed that FUSC proteins having high sequence identities with the functionally characterized FA resistance protein FusC or Fdt might be the major contributors of FA resistance. In contrast, most fungi converted FA to less toxic compounds regardless of the level of FA resistance they exhibited. Five derivatives were detected, and the detoxification of FA involved either oxidative reactions on the butyl side chain or reductive reactions on the carboxylic acid group. The production of these metabolites from widely different phyla indicates that resistance to FA by altering its structure is highly conserved. A few FA resistant saprophytic or biocontrol strains of fungi were incapable of altering FA, indicating a possible involvement of efflux transporters. Deployment of both efflux and derivatization mechanisms may be a common feature of fungal FA resistance.

Demonstration that a Klebsiella pneumoniae subsp. pneumoniae isolated from an insect (Nezara viridula) harbors a plasmid-borne type IV secretion system.

Previously, we reported the isolation of Klebsiella pneumoniae subspecies pneumoniae strain Kp 5-1 from a southern green stink bug (Nezara viridula) that is a significant pest of numerous economically important crops. We subsequently sequenced the strains whole genome. Here, we report the presence of a functional plasmid-borne type IV secretion (TFSS) system that was identified using genomic mining of the annotated genome. Comparison of the Kp 5-1 resident 186 kb plasmid (pKp 5-1) with nine other Klebsiella with plasmids of comparable size from clinical and environmental strains revealed putative TFSS with identities ranging from 70 to 99%. A primer set was designed at the pKp 5-1 region that shared homology with traC of the conjugation capable F-plasmid. The 2.4 kb amplified PCR product was cloned, sequenced, and used in hybridization experiments verify that the predicted gene was extra-chromosomally located. Based on biparental mating experimental results, a K. pneumoniae Kp 5-1 derivative transformed with the non-self-transmissible pMMB207αß (an IncQ RSF1010 derivative) mobilized the vector into the parental strain with transfer frequencies of 10-3 transconjugants/donor. Identification of a TFSS in strain Kp 5-1 is significant since in other systems the mobilization capacity is involved in dissemination of plasmids that may confer antibiotic resistance and/or the delivery of virulence proteins into host cells, and thus may have an important role in the fitness of this strain as well. This is the first report that both compared and demonstrated functionality of a plasmid-harbored TFSS in a K. pneumoniae isolated from a N. viridula.

Specific PCR Detection of Fusarium oxysporum f. sp. vasinfectum California Race 4 Based on a Unique Tfo1 Insertion Event in the PHO Gene.

A highly virulent race 4 (Cal race 4) of Fusarium oxysporum f. sp. vasinfectum was identified in California cotton fields in 2001, and has since been found in increasing numbers of fields. Cal race 4 isolates contain a unique Tfo1 transposon insertion in the PHO gene that was not found in other F. oxysporum f. sp. vasinfectum genotypes. Based on this insertion, a multiplex polymerase chain reaction method was developed to detect the Cal race 4 pathogen. A panel of F. oxysporum f. sp. vasinfectum isolates representing different vegetative compatibility groups (VCG) and DNA sequence types was assembled to test the specificity of the detection method. In all, 16 of 17 Cal race 4 isolates produced a 583-bp amplicon; the other isolate produced a 396-bp amplicon reflecting the absence of the Tfo1 insertion. This isolate was a moderately virulent pathogen among Cal race 4 isolates. In total, 80 other F. oxysporum isolates associated with cotton and 11 other formae speciales of F. oxysporum produced only the 396-bp amplicon. The method also distinguished Cal race 4 isolates from India race 4 isolates and China race 7 isolates, which did not possess the unique Tfo1 insertion but otherwise had identical DNA sequences, and all belong to VCG0114. The method is capable of detecting the pathogen directly from infected stem tissues even before external symptom appears and, thus, provides an effective tool for timely identification of infested fields and seed lots, and should help reduce dissemination of Cal race 4 in the U.S. Cotton Belt.

Genetic Diversity, Virulence, and Meloidogyne incognita Interactions of Fusarium oxysporum Isolates Causing Cotton Wilt in Georgia.

Locally severe outbreaks of Fusarium wilt of cotton (Gossypium spp.) in South Georgia raised concerns about the genotypes of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. Vegetative complementation tests and DNA sequence analysis were used to determine genetic diversity among 492 F. oxysporum f. sp. vasinfectum isolates obtained from 107 wilted plants collected from seven fields in five counties. Eight vegetative complementation groups (VCG) were found, with VCG 01117B and VCG 01121 occurring in 66% of the infected plants. The newly recognized VCG 01121 was the major VCG in Berrien County, the center of the outbreaks. All eight VCG resulted in significant increases in the percentages of wilted leaves (27 to 53%) and significant reductions in leaf weight (40 to 67%) and shoot weight (33 to 60%) after being stem punctured into Gossypium hirsutum 'Rowden'. They caused little or no significant reductions in shoot weight and height or increases in foliar symptoms and vascular browning in a soil-infestation assay. Soil infestation with Meloidogyne incognita race 3 (root-knot nematode) alone also failed to cause significant disease. When coinoculated with M. incognita race 3, all VCG caused moderate to severe wilt. Therefore, the VCG identified in this study belong to the vascular-competent pathotype, and should pose similar threats to cotton cultivars in the presence of the root-knot nematode. Use of nematode-resistant cultivars, therefore, is probably the best approach to control the disease in Georgia.

Response of AtNPR1-expressing cotton plants to Fusarium oxysporum f. sp. vasinfectum isolates.

In our earlier investigation, we had demonstrated that transgenic cotton plants expressing AtNPR1 showed significant tolerance to Fusarium oxysporum f. sp. vasinfectum, isolate 11 (Fov11) and several other pathogens. The current study was designed to further characterize the nature of the protection provided by AtNPR1 expression and its limitations. Green Fluorescent Protein-expressing Fov11 was generated and used to study the progression of the disease within the plant. The results show that the spread of the pathogen was slower in the AtNPR1-transformants compared to the wild type plants. Transcript analysis in the seedling root and hypocotyl showed that the transgenic lines are capable of launching a stronger defense response when infected with Fov11. We further confirmed that AtNPR1 transformants showed greater degree of tolerance to Fov11. However, little or no protection was observed against a related, but more virulent isolate, Fov43, and a highly virulent isolate, CA9.

FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.

Fusaric acid (FA) is a key component in virulence and symptom development in cotton during infection by Fusarium oxysporum. A putative major facilitator superfamily (MFS) transporter gene was identified downstream of the polyketide synthase gene responsible for the biosynthesis of FA in a region previously believed to be unrelated to the known FA gene cluster. Disruption of the transporter gene, designated FUBT, resulted in loss of FA secretion, decrease in FA production and a decrease in resistance to high concentrations of FA. Uptake of exogenous FA was unaffected in the disruption transformants, suggesting that FA enters the cell in Fusarium by an independent mechanism. Thus, FUBT is involved both in the extracellular transport of FA and in resistance of F. oxysporum to this non-specific toxin. A potential secondary resistance mechanism, the production of FA derivatives, was observed in FUBT deletion mutants. Molecular analysis of key biochemical processes in the production of FA could lead to future host plant resistance to Fusarium pathogens.

Relationship Between Piercing-Sucking Insect Control and Internal Lint and Seed Rot in Southeastern Cotton (Gossypium hirsutum L.).

In 1999, crop consultants scouting for stink bugs (Hemiptera spp.) in South Carolina discovered a formerly unobserved seed rot of cotton that caused yield losses ranging from 10 to 15% in certain fields. The disease has subsequently been reported in fields throughout the southeastern Cotton Belt. Externally, diseased bolls appeared undamaged; internally, green fruit contain pink to dark brown, damp, deformed lint, and necrotic seeds. In greenhouse experiments, we demonstrated transmission of the opportunistic bacterium Pantoea agglomerans by the southern green stink bug, Nezara viridula (L.). Here, green bolls were sampled from stink bug management plots (insecticide protected or nontreated) from four South Atlantic coast states (North Carolina, South Carolina, Georgia, and Florida) to determine disease incidence in the field and its association with piercing-sucking insects feeding. A logistic regression analysis of the boll damage data revealed that disease was 24 times more likely to occur (P = 0.004) in bolls collected from plots in Florida, where evidence of pest pressure was highest, than in bolls harvested in NC with the lowest detected insect pressure. Fruit from plots treated with insecticide, a treatment which reduced transmission agent numbers, were 4 times less likely to be diseased than bolls from unprotected sites (P = 0.002). Overall, punctured bolls were 125 times more likely to also have disease symptoms than nonpunctured bolls, irrespective of whether or not plots were protected with insecticides (P = 0.0001). Much of the damage to cotton bolls that is commonly attributed to stink bug feeding is likely the resulting effect of vectored pathogens.

Conversion of fusaric acid to Fusarinol by Aspergillus tubingensis: a detoxification reaction.

The fungus Fusarium oxysporum causes wilt diseases of plants and produces a potent phytotoxin fusaric acid (FA), which is also toxic to many microorganisms. An Aspergillus tubingensis strain with high tolerance to FA was isolated from soil and designated as CDRAt01. HPLC analysis of culture filtrates from A. tubingensis isolate CDRAt01 grown with the addition of FA indicated the formation of a metabolite over time that was associated with a decrease of FA. Spectral analysis and chemical synthesis confirmed the compound as 5-butyl-2-pyridinemethanol, referred to here as fusarinol. The phytotoxicity of fusarinol compared to FA was measured by comparing necrosis induced in cotton (Gossypium hirsutum L. cv. Coker 312) cotyledons. Fusarinol was significantly less phytotoxic than FA. Therefore, the A. tubingensis strain provides a novel detoxification mechanism against FA which may be utilized to control Fusarium wilt.

Nematode-resistance loci in Upland cotton genomes are associated with structural differences.

Reniform and root-knot nematode are two of the most destructive pests of conventional upland cotton, Gossypium hirsutum, L. and continue to be a major threat to cotton fiber production in semi-arid regions of the southern United States and Central America. Fortunately, naturally occurring tolerance to these nematodes has been identified in the Pima cotton species (G. barbadense) and several upland cotton varieties (G. hirsutum), which has led to a robust breeding program that has successfully introgressed and stacked these independent resistant traits into several upland cotton lineages with superior agronomic traits, e.g. BAR 32-30 and BARBREN-713. This work identifies the genomic variations of these nematode tolerant accessions by comparing their respective genomes to the susceptible, high-quality fiber producing parental line of this lineage: Phytogen 355 (PSC355). We discover several large genomic differences within marker regions that harbor putative resistance genes as well as expression mechanisms shared by the two resistant lines, with respect to the susceptible PSC355 parental line. This work emphasizes the utility of whole genome comparisons as a means of elucidating large and small nuclear differences by lineage and phenotype.  .

Enhanced resistance against Thielaviopsis basicola in transgenic cotton plants expressing Arabidopsis NPR1 gene.

Black root rot, caused by Thielaviopsis basicola, is an important disease in several crops including cotton. We studied the response of Arabidopsis NPR1 (AtNPR1)-expressing cotton lines, previously shown to be highly resistant to a diverse set of pathogens, to a challenge from T. basicola. In four different experiments, we found significant degree of tolerance in the transgenic lines to black root rot. Although transformants showed the typical root discoloration symptoms similar to the wild-type control plants following infection, their roots tended to recover faster and resumed normal growth. Better performance of transgenic plants is reflected by the fact that they have significantly higher shoot and root mass, longer shoot length, and greater number of boll-set. Transcriptional analysis of the defense response showed that the roots of AtNPR1-overexpressing transgenic plants exhibited stronger and faster induction of most of these defense-related genes, particularly PR1, thaumatin, glucanase, LOX1, and chitinase. The results obtained in this investigation provide further support for a broad-spectrum nature of the resistance conferred by overexpression of AtNPR1 gene in cotton.

Lacinilene C 7-methyl ether.

The title compound, C16H20O3 [systematic name: 1-hy-droxy-7-meth-oxy-1,6-dimethyl-4-(propan-2-yl)naphthalen-2(1H)-one], is a sesquiterpene isolated from foliar tissues of the cotton plant and is of inter-est with respect to its anti-bacterial properties. Its phenyl ring is ideally planar, and the maximum of deviation in the second ring is 0.386 (3) Å. The hy-droxy group and the methyl group are oriented in an equatorial fashion and axial, respectively, to the second ring. In the crystal, inversion dimers are formed through pairs of O-H⋯O hydrogen bonds. Weak C-H⋯O hydrogen bonds link the dimers into columns along the c axis. These columns form a crystal structure with a crystal packing factor of 0.66.

Genome sequence of Pantoea sp. strain Sc 1, an opportunistic cotton pathogen.

Pantoea is comprised of a broad spectrum of species, including plant pathogens. Here, we provide an annotated genome sequence of Pantoea sp. strain Sc 1, which was isolated from a diseased cotton boll. This research provides the first genome sequence of a bona fide Pantoea sp. insect-vectored cotton pathogen.

Phylogeny and pathogenicity of Fusarium oxysporum isolates from cottonseed imported from Australia into California for dairy cattle feed.

A unique biotype of the Fusarium wilt pathogen, Fusarium oxysporum Schlecht. f.sp. vasinfectum (Atk) Sny. & Hans., found in Australia in 1993 is favored by neutral or alkaline heavy soils and does not require plant parasitic nematodes to cause disease. This makes it a threat to 4-6 million acres of USA Upland cotton ( Gossypium hirsutum L.) that is grown on heavy alkaline soil and currently is not affected by Fusarium wilt. In 2001-2002, several shiploads of live cottonseed were imported into California for dairy cattle feed. Thirteen F. oxysporum f.sp. vasinfectum isolates and four isolates of a Fusarium spp. that resembled F. oxysporum were isolated from the imported cottonseed. The isolates, designated by an AuSeed prefix, formed four vegetative compatibility groups (VCG) all of which were incompatible with tester isolates for 18 VCGs found in the USA. Isolate AuSeed14 was vegetatively compatible with the four reference isolates of Australian biotype VCG01111. Phylogenetic analyses based on EF-1α, PHO, BT, Mat1-1, and Mat1-2 gene sequences separated the 17 seed isolates into three lineages (race A, race 3, and Fusarium spp.) with AuSeed14 clustering into race 3 lineage or race A lineage depending on the genes analyzed. Indel analysis of the EF-1α gene sequences revealed a close evolutionary relationship among AuSeed14, Australian biotype reference isolates, and the four Fusarium spp. isolates. The Australian seed isolates and the four Australian biotype reference isolates caused disease with root-dip inoculation, but not with stem-puncture inoculation. Thus, they were a vascular incompetent pathotype. In contrast, USA race A lineage isolates readily colonized vascular tissue and formed a vascular competent pathotype when introduced directly into xylem vessels. The AuSeed14 isolate was as pathogenic as the Australian biotype, and it or related isolates could cause a severe Fusarium wilt problem in USA cotton fields if they become established.

Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1.

Cotton is an economically important crop worldwide that suffers severe losses due to a wide range of fungal/bacterial pathogens and nematodes. Given its susceptibility to various pathogens, it is important to obtain a broad-spectrum resistance in cotton. Resistance to several fungal and bacterial diseases has been obtained by overexpressing the Non-expressor of Pathogenesis-Related genes-1 (NPR1) in various plant species with apparently minimal or no pleiotropic effects. We examined the efficacy of this approach in cotton by constitutive expression of the Arabidopsis (Arabidopsis thaliana) NPR1 gene. The results show that NPR1-expressing lines exhibited significant resistance to Verticillium dahliae isolate TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternata. Interestingly, the transformants also showed significant resistance to reniform nematodes. Analysis of defense-related, biochemical and molecular responses suggest that when challenged with pathogens or certain systemic acquired resistance-inducing chemicals, the transgenic lines respond to a greater degree compared to the wild-type plants. Importantly, the basal activities of the defense-related genes and enzymes in uninduced transformants were no different than those in their non-transgenic counterparts. The results provide additional evidence supporting the role of NPR1 as an important part of the plant defense system and suggest a means to achieve broad-spectrum resistance to pathogens via genetic engineering.

Complete Genome Sequence of Serratia sp. Strain CC119, Associated with Inner Cotton Boll Rot via Insect Vector Transmission.

Serratia species are Gram-negative bacteria that can infect both animals and plants. The annotated genome presented is the first for a Serratia sp. strain (called CC119) that is a cotton boll pathogen. The opportunistic strain is associated with the boll-piercing-sucking insect Creontiades signatus.

Temporal analysis of cotton boll symptoms resulting from southern green stink bug feeding and transmission of a bacterial pathogen.

The southern green stink bug, Nezara viridula (L.), is a significant pest of cotton, Gossypium hirsutum L., and is becoming an increasing challenge due to the decrease in use of broad-spectrum insecticides on the crop. The southern green stink bug can vector an opportunistic Pantoea agglomerans strain (designated Sc 1-R) into cotton bolls, resulting in infection. The appearance of stink bug damage varies, and pest managers cannot readily identify its source. This research reports a systematic depiction of green, immature boll responses at various stages of maturity (1, 2, and 3 wk post-anthesis [WPA]) to stink bug injury and to infection by the vectored cotton pathogen by demonstrating the progression of effects 1, 2, and 3 wk after exposure (WAE). When laboratory-reared adult southern green stink bug not harboring Sc 1-R deposited bacteria into greenhouse-grown bolls at 1, 2, or 3 WPA during feeding/probing, bacteria reached concentrations of 10(9), 10(9), and 10(3) colony-forming units (CFUs)/g tissue, respectively, at 3 WAE, yet caused minimal seed and lint damage regardless of the age of the bolls that were penetrated. Bolls at a maturity of 1 or 2 WPA showed similar susceptibility when exposed to stink bugs that vectored Sc 1-R. After a week of infection, seeds were salmon-pink with normal white lint and up to 10(4) CFUs/g tissue when Sc 1-R was detected. Necrosis of the entire inoculated locule(s) with a maximum Sc 1-R concentration detected at 10(8) CFUs/g tissue occurred in samples harvested 2 or 3 WAE. Conversely, seed and lint deterioration due to the transmitted opportunist into bolls exposed 3 WPA was confined to the puncture site. In summary, after a week of development, bolls were tolerant to southern green stink bug feeding/ probing damage and to nonpathogenic bacteria, but they were severely damaged when the opportunistic pathogen Sc 1-R was transmitted. At 3 WPA, the fruit was immune to the spread of the pathogen with infections confined to the puncture site.

Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein.

The terpenoid gossypol, a secondary metabolite found in the cotton plant, is synthesized by a free radical dimerization of hemigossypol. Gossypol exists as an atropisomeric mixture because of restricted rotation around the central binaphthyl bond. The dimerization of hemigossypol is regiospecific in cotton. In the case of some moco cotton, the dimerization also exhibits a high level of stereoselectivity. The mechanism that controls this stereoselective dimerization is poorly understood. In this paper, we demonstrate that a dirigent protein controls this stereoselective dimerization process. A partially purified protein preparation from cotton flower petals, which by itself is unable to convert hemigossypol to gossypol, converts hemigossypol with a 30% atropisomeric excess into (+)-gossypol when combined with an exogenous laccase, which by itself produces racemic gossypol.