Agrobacterium Tumefaciens-mediated Transformation Research Articles

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ± 0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.

Visualization of Phytophthora palmivora Infection in Oil Palm Leaflets with Fluorescent Proteins and Cell Viability Markers.

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.

In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation of Datura stramonium (Solanaceae).

Premise of the Study Datura stramonium is a pharmacologically and evolutionarily important plant species in the family Solanaceae. Stable transformation methodology of this species would be advantageous for future genetic studies.MethodsIn vitro plant regeneration and Agrobacterium tumefaciens–mediated transformation techniques were developed for D. stramonium based on methods reported for tomato. A binary vector containing pAtUBQ10::erGFP was used for transformation.ResultsWe recovered primary transformants harboring the green fluorescent protein (GFP) transgene that resulted in expression of fluorescence in all tissues analyzed. Transformants were allowed to self‐pollinate, and two of five progeny contained the GFP transgene and displayed fluorescence identical to the primary transformants.DiscussionWe have demonstrated the first stable transformation in the genus Datura. This is a key first step to study the genetic basis of traits in this evolutionarily interesting species.

Unique gene Pmhyp controlling melanization of pycnidia in Phoma medicaginis

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot of alfalfa and the model legume Medicago truncatula. Phoma medicaginis produces uninucleate conidia in melanized pycnidia and is genetically tractable through Agrobacterium tumefaciens-mediated transformation (ATMT), which can result in insertional mutants. One T-DNA-tagged mutant, P1A17 produced conidia in non-melanized (hyaline) pycnidia. Pycnidial melanization recovered if the mutant was supplemented with melanin precursors or allowed to age. DNA sequences flanking the insertion did not predict any disrupted open reading frames (ORF) unless a Coccidioides prediction algorithm was used. Pmhyp gene was expressed in the wild type, but not the mutant, and has not been annotated in any genomes, to date. Expression of two conserved genes flanking the T-DNA disrupted Pmhyp was unchanged from the wild type. Knockout of Pmhyp strain displayed same cultural phenotype (non-melanized pycnidia). Complementation of Pmhyp strains with wild type PmHYP partially recovered pycnidial melanization. Both knockout and complementation transformants were confirmed using RT-PCR and southern blot analysis. Taken together, PmHYP appears to be a novel regulator of pycnidium specific melanization.

Plant regeneration and Agrobacterium-mediated transformation of the miracle tree Neolamarckia cadamba

Neolamarckia cadamba is a miracle tree species with considerable economic potential used as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish an efficient protocol of plant regeneration and Agrobacterium-mediated genetic transformation for N. cadamba. Greenish callus were induced from different type explants when cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) and kinetin (KT). The callus could differentiate into somatic embryos or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. It was found that MS medium supplemented with 2 mg/L TDZ, 0.2 mg/L KT provided the most suitable medium for leaf explants to induce adventitious shoot, delivering a maximum regeneration frequency with a mean of 8.2 shoots per explant. The micro-shoots developed roots in MS supplemented with 0.05 mg/L α-naphthaleneacetic acid (NAA), and could be successfully acclimatized to a greenhouse with more than 90% survival. Based on this regeneration protocol, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for N. cadamba. Genetic transformation was obtained through the infection of leaf with A. tumefaciens strain GV3101 harboring a modified binary vector pCAMBIA1305 containing the marker genes β-glucuronidase (GUS) and fluorescent protein YFP. The presence of GUS in transformed plants was confirmed using PCR and histological stain, while the expression of YFP was confirmed through fluorescent microscopy and RT-PCR. Out of 276 putative transformants, 15 plants were found to be positive transgenic. Nevertheless, by adopting this protocol, positive transformed plants could be obtained within three months.

Enhancement of Torularhodin Production in Rhodosporidium toruloides by Agrobacterium tumefaciens-Mediated Transformation and Culture Condition Optimization.

Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box-Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.

An Enzymolysis-Assisted Agrobacterium tumefaciens-Mediated Transformation Method for the Yeast-Like Cells of Tremella fuciformis

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

Cloning and Function Verification of gpd Promoter from Hypoxylon sp.

In order to construct the transformation system and study the gene function of Hypoxylon sp.,a highly active promoter element was necessary for the transformation system. In this experiment,the gpd gene and the sequence of about 1000 bp upstream of the gene were cloned by PCR. The results of Neural Network Promoter Prediction,Signal Scan Search Request and PLANTCAREA database analysis showed that the upstream region of the gpd gene contained not only the characteristic core required by the promoter,such as TATA-box,CAAT-box,G-box,GC-box,CT-rich,etc,but also contained a high score transcription start site. To further verify the promoter function of this sequence,the gpd promoter element was ligated with the enhanced green fluorescent protein gene(egfp)and hygromycin gene(hph)to construct a fusion expression vector,which was transformed by Agrobacterium tumefaciens-mediated transformation. Hygromycin resistance screening,green fluorescent protein detection results showed that the gpd promoter of Hypoxylon sp. successfully drove the expression of hygromycin resistance gene and enhanced green fluorescent protein gene,and Agrobacterium tumefaciens-mediated transformation was successful. It laid a foundation for the study of exogenous gene expression and gene function in Hypoxylon sp.

Transient Expression Assay in Strawberry Fruits.

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar 'Sweet Charlie'. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites.

The revolutionary discovery of penicillin only marks the start of our exploration for valuable fungal natural products. Advanced genome sequencing technologies have translated the fungal genome into a huge reservoir of “recipes” – biosynthetic gene clusters (BGCs) – for biosynthesis. Studying complex fungal genetics demands specific gene manipulation strategies. This review summarizes the current progress in efficient gene targeting in fungal cells and heterologous expression systems for expressing fungal BGCs of fungal secondary metabolites.

Efficient agrobacterium-mediated transformation of Shiraia bambusicola and activation of a specific transcription factor for hypocrellin production

Shiraia bambusicola, as a well-known medicinal and edible macro-fungus, is generally located on the branches of bamboo and plays an important role in herbal treatment. Furthermore, the natural products from its fruiting bodies, hypocrellins, are attracting increasing amounts of attention due to their notable bioactivities. In this report, we focused on constructing a fast and universal transgenic system for S. bambusicola, which could easily regulate the biosynthesis of hypocrellins by altering gene expression. By the consideration of the broadened application of the mycelial transformation, it was determined to implement the Agrobacterium tumefaciens-mediated transformation (ATMT) technique, and a transcription factor (SbTF1) was employed to activate the downstream genes in different producing strains. After real-time polymerase chain reaction analysis, gene SbTF1 was verified to be overexpressed in all of the transgenic strains, and the yield of hypocrellins was significantly improved in strain CNU103846. To confirm the regulated function, the transcript levels of the downstream genes and the corresponding products at different stages were screened. In this study, we developed a stable and easily available assay for genetic transformation of S. bambusicola and employed the method to demonstrate that one transcription factor SbTF1 could effectively increase the yield of the final products.

Efficient Transformation of the White Jelly Mushroom Tremella fuciformis (Tremellomycetes) and Its Companion Fungus Annulohypoxylon stygium (Ascomycetes) Mediated by Agrobacterium tumefaciens.

Tremella fuciformis is an edible white jelly mushroom with medicinal qualities. The formation of T. fuciformis fruiting bodies is highly dependent on the presence of Annulohypoxylon stygium under natural conditions and during artificial cultivation. A lack of efficient transformation systems restricts the ability of researchers to functionally characterize the genes in these two interacting fungi. In this study, we tested the utility of the Agrobacterium tumefaciens-mediated transformation of T. fuciformis and A. stygium protoplasts. A. tumefaciens strain EHA105 cells harboring the pTrGEH plasmid, which contains genes for enhanced green fluorescent protein (egfp) and hygromycin B phosphotransferase (hph), was co-cultured with T. fuciformis protoplasts. Meanwhile, EHA105 cells harboring the pAnGRH plasmid, which contains the red fluorescent protein (rfp) and hph genes, was co-cultured with A. stygium protoplasts. The egfp, rfp, and hph genes were under the control of the promoter for gpd, which encodes glyceraldehyde-3-phosphate dehydrogenase. Optimal co-cultivation was achieved with a 1:1 mixture of bacteria (OD600 0.4-0.6) and fungal protoplasts (105/mL) incubated at 25°C in a medium containing 200 μM acetosyringone. The subsequent selection on hygromycin B-containing medium yielded 45 and 187 stable transformants per 105 protoplasts for T. fuciformis and A. stygium, respectively. The integration of the transformed DNA into the two fungal genomes was confirmed by polymerase chain reaction (PCR), Southern blot analysis, fluorescence imaging, and a quantitative real-time PCR. All results confirmed the feasibility of our transformation approach, which may facilitate future functional analyses of T. fuciformis and A. stygium genes.

Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi.

We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.

A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042.

Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplastmediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

Cotton Hairy Root Culture as an Alternative Tool for Cotton Functional Genomics.

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In certain cases, A. rhizogenes-induced hairy root culture has been a suitable molecular tool for functional analyses of genes and promoters for plants that are difficult to regenerate by A. tumefaciens-mediated transformation. Similarly, A. rhizogenes-induced hairy root cultures are an alternative tool for cotton functional genomics. In this chapter, the advantages and disadvantages of using A. rhizogenes-induced cotton hairy root culture over A. tumefaciens-mediated cotton transformation are discussed. The procedures for transformation, generation, selection, and molecular analyses of transgenic cotton hairy roots are introduced by describing the functional analysis of a cotton promoter in cotton hairy roots generated by A. rhizogenes-mediated transformation.

Susceptibility of Maize Mesocotyl Culture to Agrobacterium Transformation and Its in vitro Regeneration

The capacity of mesocotyls to agrobacterially transform four maize lines 4766, RB-179, L-1, L-2 and the conditions for their in vitro regeneration have been studied. Regeneration of maize mesocotyls was observed in media containing various auxins and cytokinins combinations. The efficiency of the process depended on the qualitative and quantitative compositions of growth regulators. The frequency of regenerated explants and the average number of shoots that form on one explant increased with increased cytokinin 6‑BAP and defoliant Dropp (thidiazuron) concentrations in the medium. 6-BAP (3–5 mg/L) and Dropp (3 mg/L) in combination with auxins (IAA, 0.5 mg/L and 2,4-D, 2 mg/L) were shown to be most effective for the mesocotyl regeneration. The number of regenerated explants reached 59.0%, whereas the average number of shoots per explant was 7.5 after 6 weeks of cultivation. The rooting and growing of regenerated shoots was successfully carried out on MS medium supplemented with 0.2 mg/L IBA. The capacity of the mesocotyl was determined by Agrobacterium tumefaciens–mediated transformation (ATMT) with an AGL0 strain containing the pVecActI-GUS genetic construction encoding the reporter gene uidA under the control of the promoter of rice actin ActI gene and the selective hptII hygromycin resistance gene. PCR analysis demonstrated that few regenerants with resistance to the selective agent contained the insertion of the hptII antibiotic-resistance gene in their genomes. Thus, it was shown that maize mesocotyls can be used as explants for agrobacterial transformation.

Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat

BackgroundRecently, the CRISPR/Cas9 system has been widely used to precisely edit plant genomes. Due to the difficulty in Agrobacterium-mediated genetic transformation of wheat, the reported applications in CRISPR/Cas9 system were all based on the biolistic transformation.ResultsIn the present study, we efficiently applied targeted mutagenesis in common wheat (Triticum aestivum L.) protoplasts and transgenic T0 plants using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Seven target sites in three genes (Pinb, waxy and DA1) were selected to construct individual expression vectors. The activities of the sgRNAs were evaluated by transforming the constructed vectors into wheat protoplasts. Mutations in the targets were detected by Illumina sequencing. Genome editing, including insertions or deletions at the target sites, was found in the wheat protoplast cells. The highest mutation efficiency was 6.8% in the DA1 gene. The CRISPR/Cas9 binary vector targeting the DA1 gene was then transformed into common wheat plants by Agrobacterium tumefaciens-mediated transformation, resulting in efficient target gene editing in the T0 generation. Thirteen mutant lines were generated, and the mutation efficiency was 54.17%. Mutations were found in the A and B genomes of the transgenic plants but not in the D genome. In addition, off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences.ConclusionsOur results showed that our Agrobacterium-mediated CRISPR/Cas9 system can be used for targeted mutations and facilitated wheat genetic improvement.

Enhanced Agrobacterium-mediated transformation efficiency of banana cultivar Grand Naine by reducing oxidative stress

Efficient protocols were developed for somatic embryogenesis (SE) and recovery of genetic transformants of banana cultivar Grand Naine. The efficiency of embryogenic callus formation was enhanced up to 14% by spraying 2, 4-dichlorophenoxyacetic acid (20 mg/l) on immature male flower buds (IMFBs) before inoculation of explants (immature male flowers) on the callus induction medium (CIM). Embryogenic cell suspension (ECS) derived from callus was used for Agrobacterium tumefaciens-mediated transformation harboring β-glucuronidase (GUS) as a reporter and hygromycin phosphotransferase (hpt II) as the selection marker genes. Four antioxidants (melatonin, quercetin, gallic acid, and α-tocopherol) were tested during and after Agrobacterium infection of ECS to reduce the oxidative stress. Melatonin boosted up nearly 1.5-fold [72.3 ± 7.5 plantlets/ml settled cell volume (SCV)] higher transformation efficiency as compared to without antioxidant treated (46.7 ± 2.5 plantlets/ml SCV) culture. Scanning electron microscopy (SEM) revealed that melatonin reinforced the bacterial attachment to the plant cell and also maintained surface morphology of the cells. The expression of the reporter gene in putatively transformed ECS and different tissues has been demonstrated by histochemical GUS assay. The transgenic plants were confirmed by PCR for the presence of hpt II and GUS amplicon. The optimized efficient protocols pave the way to generate the high number of transgenic lines with desirable traits in important commercial banana cultivar Grand Naine.

ATMT transformation efficiencies with native promoters in Botryosphaeria kuwatsukai causing ring rot disease in pear.

Botryosphaeria kuwatsukai is an important fungal pathogen affecting pear fruits. However, infection processes of this fungus are still unclear. This study seeks to develop the fungal transformation of B. kuwatsukai by Agrobacterium tumefaciens-mediated transformation (ATMT), assess the reliability of appropriate vectors and examine the infection processes in vitro using a GFP labeled strain of B. kuwatsukai. To establish a highly effective transformation system in B. kuwatsukai, binary vectors containing various lengths of H3 promoters and TEF promoters fused with GFP and hygromycin B resistance gene cassettes were constructed. These cassettes were integrated into the genomic DNA of B. kuwatsukai with high transformation frequency by the ATMT method. Transformants showed strong expression of GFP and hygromycin B resistance genes in cells. Furthermore, we investigated if native promoters are more suitable to govern marker genes than other general promoters used in other filamentous fungi. The results obtained herein demonstrate that the vectors constructed in this study can be utilized with high transformation rate. Microscopic examinations also reveal that fungal hyphae undergo morphological changes during the infection process resulting in biotrophic stage of infected host cells. Our results provide genetic insights to further explore the infection processes of B. kuwatsukai.

Genetic modification of asexual Epichlo\xeb endophytes with the perA gene for peramine biosynthesis

Development of grass–endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.