A newly evolved rice-specific gene JAUP1 regulates jasmonate biosynthesis and signalling to promote root development and multi-stress tolerance.

Root architecture and function are critical for plants to secure water and nutrient supply from the soil, but environmental stresses alter root development. The phytohormone jasmonic acid (JA) regulates plant growth and responses to wounding and other stresses, but its role in root development for adaptation to environmental challenges had not been well investigated. We discovered a novel JA Upregulated Protein 1 gene (JAUP1) that has recently evolved in rice and is specific to modern rice accessions. JAUP1 regulates a self-perpetuating feed-forward loop to activate the expression of genes involved in JA biosynthesis and signalling that confers tolerance to abiotic stresses and regulates auxin-dependent root development. Ectopic expression of JAUP1 alleviates abscisic acid- and salt-mediated suppression of lateral root (LR) growth. JAUP1 is primarily expressed in the root cap and epidermal cells (EPCs) that protect the meristematic stem cells and emerging LRs. Wound-activated JA/JAUP1 signalling promotes crosstalk between the root cap of LR and parental root EPCs, as well as induces cell wall remodelling in EPCs overlaying the emerging LR, thereby facilitating LR emergence even under ABA-suppressive conditions. Elevated expression of JAUP1 in transgenic rice or natural rice accessions enhances abiotic stress tolerance and reduces grain yield loss under a limited water supply. We reveal a hitherto unappreciated role for wound-induced JA in LR development under abiotic stress and suggest that JAUP1 can be used in biotechnology and as a molecular marker for breeding rice adapted to extreme environmental challenges and for the conservation of water resources.

A Large-Scale Hydroponic Evaluation of Rice Mutants for Pythium Resistance.

Rice is a major staple crop worldwide. However, the occurrence of rice diseases during cultivation poses a significant challenge to achieving optimal yields. Among the major pathogens, Pythium species, which cause seedling blight, are of particular concern. Pythium infects rice roots through zoospores, mycelia, or oospores, leading to root rot, stunting, yellowing, and ultimately seedling damping-off. While many disease resistance-related genes have been reported in rice, only very limited research has been associated with resistance to Pythium infection. In this study, we aimed to establish a rapid screening system to identify rice lines that are resistant or susceptible to Pythium pathogen in rice nurseries. We conducted evaluations on important factors, including virulence, inoculation method, seed soaking period, and the measurement of disease severity. As a result, we successfully developed a screening system that allows for high-throughput and rapid screening of the Taiwan Rice Insertional Mutant (TRIM) library for mutant lines exhibiting resistance to P. arrhenomanes. Furthermore, we identified a slightly resistant TRIM line and explored potential genes encoding endglucanase-1 precursor and malonyl-CoA decarboxylase that may be involved in conferring resistance to P. arrhenomanes.

Endophytic Bacterium Lysobacter firmicutimachus Strain 5-7 Is a Promising Biocontrol Agent Against Rice Seedling Disease Caused by Pythium arrhenomanes in Nursery Trays.

Rice root rot disease caused by Pythium spp. is a highly destructive disease in rice nurseries. Biocontrol with endophytic bacteria was developed in this study to control rice seedling diseases. An in planta screening assay revealed that two bacterial endophytes, strains 5-7 and 6-4, displayed strong protection of rice seedlings from attack by Pythium arrhenomanes. Phylogenetic analysis indicated that strain 5-7 is Lysobacter firmicutimachus, while strain 6-4 belongs to the Kitasatospora genus. To quickly evaluate the disease severity of the root system damaged by Pythium spp. in nursery trays, a root surface area measurement assay was developed. By using this measurement, the control efficacy in nursery trays was evaluated, and L. firmicutimachus 5-7 showed promising biocontrol activity against Pythium disease. In a field trial, the two endophytes exhibited significant disease control efficacy on rice brown spot disease caused by Bipolaris oryzae naturally occurring in a commercial nursery field. The two endophytes exhibited multiple enzymatic activities and broad-spectrum antagonistic activities against multiple rice pathogens. The two endophytes colonized the root surface and inside of the root. L. firmicutimachus 5-7 primarily colonized the intercellular space and aerenchyma. Antibiosis is the major mechanism used by strain 5-7 to cause Bipolaris hyphal swelling and inhibit Pythium zoospore germination and sporangium formation, while a hyperparasitism-like phenomenon was found in the interaction of strain 6-4 with Pythium and Bipolaris hyphae. In conclusion, we report the promising biocontrol agent L. firmicutimachus 5-7 and the potential biocontrol agent Kitasatospora sp. 6-4 for disease control of rice seedlings in commercial nursery trays and their possible mechanisms of action.

The G protein subunit α1, CaGα1, mediates ethylene sensing of mango anthracnose pathogen Colletotrichum asianum to regulate fungal development and virulence and mediates surface sensing for spore germination.

Mango is an important tropic fruit, but its production is highly restricted by anthracnose diseases. Mango anthracnose development is related to the fruit-ripening hormone ethylene, but how the pathogen senses ethylene and affects the infection remains largely unknown. In this study, mango pathogen Colletotrichum asianum strain TYC-2 was shown to sense ethylene to enhance spore germination, appressorium formation and virulence. Upon further analysis of ethylene sensing signaling, three histidine kinase genes (CaHKs) and a G-protein gene (CaGα1) were functionally characterized. Ethylene upregulated the expression of the three CaHKs but had no influence on CaGα1 expression. No function in ethylene sensing was identified for the three CaHKs. Ethylene enhanced spore germination and multiple appressorium formation of the wild-type TYC-2 but not CaGα1 mutants. TYC-2 has extremely low germination in water, where self-inhibition may play a role in ethylene sensing via CaGα1 signaling. Self-inhibitors extracted from TYC-2 inhibited spore germination of TYC-2 and CaGα1 mutants, but ethylene could not rescue the inhibition, indicating that the self-inhibition was not mediated by CaGα1 and had no interactions with ethylene. Interestingly, spore germination of CaGα1 mutants was significantly enhanced in water on hydrophobic but not hydrophilic surfaces, suggesting that CaGα1 is involved in surface sensing. In the pathogenicity assay, CaGα1 mutants showed less virulence with delayed germination and little appressorium formation at early infection on mango leaves and fruit. Transcriptome and qRT-PCR analyses identified several pathogenicity-related genes regulated by ethylene, indicating that ethylene may regulate TYC-2 virulence partially by regulating the expression of these genes.

First report of Pythium aristosporum causing root rot on rice seedling in Taiwan.

Rice (Oryza sativa L.) is the principle staple crops in the World and its production can be severely damaged by Pythium species. Several Pythium species including P. afertile, P. arrhenomanes, P. dissotocum, P. elongatum, P. spinosum, have been recorded to cause rice seedling root rot in Taiwan (List of Plant Diseases in Taiwan edited by Tzean et al., 2019). During the survey of rice seedling diseases, we identified a new species of Pythium that causes seedling root rot on rice in commercial nursery trays in two nursery fields in 2019 in Taichung, Taiwan. Stunting and root rot symptom were found on the affected plants and up to 20% seedlings in a nursery tray showed similar symptoms. To isolate the pathogen, symptomatic roots were surface sterilized with 75% ethanol for 1 min and rinsed in sterile water. The margin of lesion was cut off, placed on 1.5% water agar and incubated at 28 ℃. After 24 h, the hyphal tips of a white colony growing from the diseased region were transferred to potato dextrose agar (PDA) medium. Koch's postulates were fulfilled by inoculating the germinated rice seeds with mycelia. Rice seeds of O. sativa var. Tainan11 (TN11) were treated with 75% ethanol and then 1.2% NaOCl for 15 min. The sterilized seeds were soaked in sterile water under dark condition for 3 days and the water was replaced every day. Five of the pre-germinated seeds with 2~5 mm embryonic shoot were placed in a sterile petri-dish and inoculated with 3-ml mycelial suspension (OD600 = 0.045) prepared by blending the mycelia of a 3-days PDA culture using an Oster 10 speed blender 6640 (Oster, USA). The seeds-mycelia were then covered with sterilized soil mixture of Akadama soil and rice husk (1:1, volume to volume) and incubated in a growth chamber at 28 ℃. Seven days post-inoculation, the inoculated seedlings showed stunting with short and necrotic roots (Fig. S1). The pathogen was reisolated from the diseased seedlings and identified with morphology and molecular methods. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia and zoospore (Chamswarng and Cook 1985). Globose oogonia with multiple antheridia (1-5 per oogonium), inflated filamentous sporangia, vesicle with abundant zoospores, main hypha with up to 6.57 µm wide and mature aplerotic oospores with diameter 24.35-30.81 µm (average= 27.22 µm; n=20) were observed (Fig. S1) that are similar to the descriptions for P. aristosporum (van der Plaats-Niterink 1981). Genomic DNA was extracted with CTAB method (Wang and White 1997) and the sequences of the internal transcribed spacer (ITS) region and gene region of ß-tubulin (tub) and cytochrome c oxidase subunit II (cox II) were amplified with published primers (Villa et al., 2006). The obtained sequences were submitted to GenBank (accession nos: OL701302 (ITS), OL763269 (tub), and OL763270 (cox II); Fig. S2). Phylogenetic relationships between this Pythium pathogen and other 55 Pythium isolates, including the type species of P. aristosporum (ATCC11101), were conducted with the concatenated sequences of tub and cox II and analyzed by Bayesian interference (Fig. S3). Based on the tree built with tub and cox II sequences, this pathogen was identified as P. aristosporum that has not been reported in rice and other plants in Taiwan. It was observed in laboratory assays that this pathogen caused significant root-rot symptoms on several major rice varieties grown in Taiwan, including TN11, Tainung67 and Kaoshiung139. It may potentially cause severe crop loss in rice production, especially in nurseries. This identification provides important information on rice disease management.

Comparative Genomics of Three Colletotrichum scovillei Strains and Genetic Analysis Revealed Genes Involved in Fungal Growth and Virulence on Chili Pepper.

Colletotrichum scovillei causes anthracnose of chili pepper in many countries. Three strains of this pathogen, Coll-524, Coll-153, and Coll-365, show varied virulence on chili pepper. Among the three strains, Coll-365 showed significant defects in growth and virulence. To decipher the genetic variations among these strains and identify genes contributing to growth and virulence, comparative genomic analysis and gene transformation to show gene function were applied in this study. Compared to Coll-524, Coll-153, and Coll-365 had numerous gene losses including 32 candidate effector genes that are mainly exist in acutatum species complex. A cluster of 14 genes in a 34-kb genomic fragment was lost in Coll-365. Through gene transformation, three genes in the 34-kb fragment were identified to have functions in growth and/or virulence of C. scovillei. CsPLAA encoding a phospholipase A2-activating protein enhanced the growth of Coll-365. A combination of CsPLAA with one transcription factor CsBZTF and one C6 zinc finger domain-containing protein CsCZCP was found to enhance the pathogenicity of Coll-365. Introduction of CsGIP, which encodes a hypothetical protein, into Coll-365 caused a reduction in the germination rate of Coll-365. In conclusion, the highest virulent strain Coll-524 had more genes and encoded more pathogenicity related proteins and transposable elements than the other two strains, which may contribute to the high virulence of Coll-524. In addition, the absence of the 34-kb fragment plays a critical role in the defects of growth and virulence of strain Coll-365.

Brown Root Rot Disease of Phyllanthus myrtifolius: The Causal Agent and Two Potential Biological Control Agents.

Brown root rot (BRR), caused by Phellinus noxius (Corner) G. Cunningham, occurs on over 200 species of plants, especially woody trees and shrubs. Ceylon myrtle (Phyllanthus myrtifolius [Wight] Müll.Arg.), a common hedge plant, was recently observed to be infected with BRR. Disease diagnosis was performed by completing Koch's postulates, and Ceylon myrtle was confirmed to be a new host of P. noxius. Typical symptoms of BRR were observed, including reduction in leaf size, dieback of branches, and suspended growth of young leaves. A disease severity index was used to quantify BRR in this study. Compared with Malabar chestnut, Ceylon myrtle was relatively resistant to BRR. Surprisingly, phylogenetic analysis of the ITS and 28S sequences revealed that isolates identified as P. noxius from Taiwan and many other countries were clustered in the same clade but separate from the clade comprising isolates from China, which were designated Pyrrhoderma noxium based on P. noxius. Therefore, to temporarily distinguish these pathogens, the former clade was designated GPN (global P. noxius), whereas the latter clade was designated CPN (China Py. noxium). In biocontrol assays, Streptomyces padanus and Bacillus sp. were selected for BRR control of Ceylon myrtle. Disease severity was reduced from 0.51 to 0.37 by S. padanus and to 0.14 by Bacillus sp. in greenhouse trials. In addition, the two biocontrol agents, especially S. padanus, exhibited good growth-promoting effects on cuttings of Ceylon myrtle. With these double advantages, S. padanus and Bacillus sp. have great potential to control BRR in practical applications.

Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

CaNRT2.1 Is Required for Nitrate but Not Nitrite Uptake in Chili Pepper Pathogen Colletotrichum acutatum.

Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by Colletotrichum acutatum. Despite the impact of C. acutatum on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of C. acutatum on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including CaGpiP1 encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, CaNRT2.1 encoding a membrane-bound nitrate/nitrite transporter, and CaRQH1 encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the CaHP1 gene encoding a hypothetical protein. Functional characterization of CaGpiP1, CaNRT2.1, and CaHP1 by targeted gene disruption and bioassays indicated that CaNRT2.1 is responsible for the growth-defective phenotype of B7. Both B7 and CaNRT2.1 mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to CaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of CaGpiP1 and CaHP1 in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in C. acutatum. Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of C. acutatum.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

Induction of apoptosis and ganoderic acid biosynthesis by cAMP signaling in Ganoderma lucidum.

Apoptosis is an essential physiological process that controls many important biological functions. However, apoptosis signaling in relation to secondary metabolite biosynthesis in plants and fungi remains a mystery. The fungus Ganoderma lucidum is a popular herbal medicine worldwide, but the biosynthetic regulation of its active ingredients (ganoderic acids, GAs) is poorly understood. We investigated the role of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in fungal apoptosis and GA biosynthesis in G. lucidum. Two phosphodiesterase inhibitors (caffeine and 3-isobutyl-1-methylxanthine, IBMX) and an adenylate cyclase activator (sodium fluoride, NaF) were used to increase intracellular cAMP levels. Fungal apoptosis was identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and a condensed nuclear morphology. Our results showed that GA production and fungal apoptosis were induced when the mycelium was treated with NaF, caffeine, or cAMP/IBMX. Downregulation of squalene synthase and lanosterol synthase gene expression by cAMP was detected in the presence of these chemicals, which indicates that these two genes are not critical for GA induction. Transcriptome analysis indicated that mitochondria might play an important role in cAMP-induced apoptosis and GA biosynthesis. To the best of our knowledge, this is the first report to reveal that cAMP signaling induces apoptosis and secondary metabolite production in fungi.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice.

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

YAP1 homologue-mediated redox sensing is crucial for a successful infection by Monilinia fructicola.

Monilinia fructicola (G. Winter) Honey is a devastating pathogen on Rosaceae which causes blossom blight and fruit rot. Only a few studies related to the plant-pathogen interaction have been published and there is limited knowledge on the relationship between oxidative stress and successful infection in M. fructicola. In this study, we cloned and characterized a redox-responsive transcription factor MFAP1, a YAP1 homologue. MfAP1-silenced strains were generated by polyethylene glycol-mediated protoplast transformation or Agrobacterium T-DNA-mediated transformation. Pathogenicity assay demonstrated that MfAP1-silenced strains caused smaller lesions on rose and peach petals. Transformants carrying extra copies of MfAP1, driven by the native promoter, were generated for MfAP1 overexpression. Interestingly, MfAP1-overexpressing strains also caused smaller lesions on rose petals. Strains carrying two copies of MfAP1 accumulated reactive oxygen species (ROS) at higher levels and exhibited delayed accumulation of MfAP1 transcripts compared with the wild-type during pathogenesis. By the analysis of ROS production and the expression patterns of redox- and virulence-related genes in the wild-type strain and an MfAP1-overexpressing strain, we found that the M. fructicola wild-type strain responded to oxidative stress at the infection site, activated the expression of MfAP1 and up-regulated the genes required for ROS detoxification and fungal virulence. In contrast, MfAP1 expression in the MfAP1-overexpressing strain was suppressed after the induction of a strong oxidative burst at the infection site, altering the expression of ROS detoxification and virulence-related genes. Our results highlight the importance of MfAP1 and ROS accumulation in the successful infection of M. fructicola.

Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola.

Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the ß-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus-M. fructicola interactions.

A novel approach to enhancing ganoderic acid production by Ganoderma lucidum using apoptosis induction.

Ganoderma lucidum is one of most widely used herbal medicine and functional food in Asia, and ganoderic acids (GAs) are its active ingredients. Regulation of GA biosynthesis and enhancing GA production are critical to using G. lucidum as a medicine. However, regulation of GA biosynthesis by various signaling remains poorly understood. This study investigated the role of apoptosis signaling on GA biosynthesis and presented a novel approach, namely apoptosis induction, to increasing GA production. Aspirin was able to induce cell apoptosis in G. lucidum, which was identified by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling assay positive staining and a condensed nuclear morphology. The maximum induction of lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24, GA24) production and total GA production by aspirin were 2.7-fold and 2.8-fold, respectively, after 1 day. Significantly lower levels of GA 24 and total GAs were obtained after regular fungal culture for 1.5 months. ROS accumulation and phosphorylation of Hog-1 kinase, a putative homolog of MAPK p38 in mammals, occurred after aspirin treatment indicating that both factors may be involved in GA biosynthetic regulation. However, aspirin also reduced expression of the squalene synthase and lanosterol synthase coding genes, suggesting that these genes are not critical for GA induction. To the best of our knowledge, this is the first report showing that GA biosynthesis is linked to fungal apoptosis and provides a new approach to enhancing secondary metabolite production in fungi.

Enhanced production of ganoderic acids and cytotoxicity of Ganoderma lucidum using solid-medium culture.

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.

Mutations of ß-tubulin codon 198 or 200 indicate thiabendazole resistance among isolates of Penicillium digitatum collected from citrus in Taiwan.

Penicillium digitatum causes green mold on citrus, resulting in severe postharvest fruit decay and economic losses in many citrus-producing areas of the world. Forty isolates of P. digitatum were cultured from citrus groves, packinghouses, and local markets in Taiwan, and assessed quantitatively for their sensitivity to thiabendazole (TBZ) fungicide. Sensitivity assays using a 96-well microtiter plate revealed that, of 40 isolates examined, only one isolate collected from fruit produced in Taiwan and two isolates from Florida-imported citrus fruit were sensitive to TBZ. The concentration of TBZ causing a 50% growth reduction (EC(50)) was less than 1 µg/mL. The remaining 37 isolates could tolerate high concentrations of TBZ, with an EC(50) greater than 80 µg/mL. Overall, more than 97% of P. digitatum isolates tested in Taiwan were found to be resistant to TBZ. In vitro assays also revealed the ineffectiveness of TBZ for controlling a TBZ-resistant isolate on sweet oranges. A sequence analysis of ß-tubulin genes revealed that all TBZ-resistant isolates displayed a single transversion point mutation, resulting in a change at either amino acid 198 (glutamic acid→glutamine) or 200 (phenylalanine→tyrosine). The repetitive use of a single fungicide over several decades has favored the selection and dominance of TBZ-resistant isolates of P. digitatum.

Overexpression of a redox-regulated cutinase gene, MfCUT1, increases virulence of the brown rot pathogen Monilinia fructicola on Prunus spp.

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H(2)O(2) enhanced MfCUT1 gene expression. A beta-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach.

To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.

Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae.

Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.