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Journal of Fungi (Basel, Switzerland) Sep 2022Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To...
Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from . In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (-) revealed the corresponding aldehyde formation in 14 cases (-). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, ) and vanillic acid (). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.
PubMed: 36294566
DOI: 10.3390/jof8101001 -
Antibiotics (Basel, Switzerland) Sep 2022Genome mining has become an important tool for discovering new natural products and identifying the cryptic biosynthesis gene clusters. Here, we utilized the...
Genome mining has become an important tool for discovering new natural products and identifying the cryptic biosynthesis gene clusters. Here, we utilized the flavin-dependent halogenase GedL as the probe in combination with characteristic halogen isotope patterns to mine new halogenated secondary metabolites from our in-house fungal database. As a result, two pairs of atropisomers, pestalachlorides A1a (1a)/A1b (1b) and A2a (2a)/A2b (2b), along with known compounds pestalachloride A (3) and SB87-H (4), were identified from Pestalotiopsis rhododendri LF-19-12. A plausible biosynthetic assembly line for pestalachlorides involving a putative free-standing phenol flavin-dependent halogenase was proposed based on bioinformatics analysis. Pestalachlorides exhibited antibacterial activity against sensitive and drug-resistant S. aureus and E. faecium with MIC values ranging from 4 μg/mL to 32 μg/mL. This study indicates that halogenase-targeted genome mining is an efficient strategy for discovering halogenated compounds and their corresponding halogenases.
PubMed: 36289962
DOI: 10.3390/antibiotics11101304 -
Plant Disease Oct 2022Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China...
Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China (Cai et al. 2015). From May to July in 2022, symptoms of leaf spot were observed on R. delavayi over a wide portion of the Baili Azalea Forest Area (N 27°10'-27°20', E 105°04'-106°04'), Guizhou Province, China. About 500 plants were surveyed and the incidence of leaf spot on R. delavayi leaves was 20 to 30%, significantly reducing their ornamental and economic value. The affected leaves had irregular, dark brown lesions with a clear blackish brown boundary and black conidiomata in a grayish center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GUDJ 61, GUDJ 62, GUDJ 63) that produced identical in morphology were obtained. The isolate GUDJ 61 was used for further study. Colonies on PDA grew velvety white on the upper surface and light yellow on the lower surface. Conidia were 5-celled, spindle- to ellipsoid-shaped, straight or slightly curved, 4-septate, and measured 39.0 ± 3.7 × 10.4 ± 0.79 µm (n=50). The morphological features were consistent with the description of Pestalotiopsis scoparia Maharachch., K.D. Hyde & Crous, (Maharachchikumbura et al. 2014). The pathogen was confirmed to be P. scoparia by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), and the partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b, and EF1-728F/EF-2, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers OP048045 (ITS), OP058111 (TUB) and OP058114 (TEF), respectively. BLAST searches of the sequences revealed 99% (549/552 nt), 99% (711/714 nt), and 82% (130/158 nt) homology with those of P. scoparia CBS 176.25T form GenBank (KM199330, KM199393, and KM199478), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUDJ 61 in a well-supported cluster with P. scoparia. The pathogen was thus identified as P. scoparia based on the morphological characterization and molecular analyses. The pathogenicity of GUDJ 61was tested through a pot assay. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GUDJ 61 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times (Fang 2007). After 12 days, the treated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. delavayi caused by P. scoparia in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. delavayi.
PubMed: 36265150
DOI: 10.1094/PDIS-08-22-1896-PDN -
Transcriptomic Analysis to Unravel Potential Pathways and Genes Involved in Pecan () Resistance to .International Journal of Molecular... Oct 2022Fruit black spot (FBS), a fungal disease of pecan (Carya illinoinensis (Wangenh) K. Koch) caused by the pathogen Pestalotiopsis microspora, is a serious disease and...
Fruit black spot (FBS), a fungal disease of pecan (Carya illinoinensis (Wangenh) K. Koch) caused by the pathogen Pestalotiopsis microspora, is a serious disease and poses a critical threat to pecan yield and quality. However, the details of pecan responses to FBS infection at the transcriptional level remain to be elucidated. In present study, we used RNA-Seq to analyze differential gene expression in three pecan cultivars with varied resistance to FBS infection: Xinxuan-4 (X4), Mahan (M), and Wichita (W), which were categorized as having low, mild, and high susceptibility to FBS, respectively. Nine RNA-Seq libraries were constructed, comprising a total of 58.56 Gb of high-quality bases, and 2420, 4380, and 8754 differentially expressed genes (DEGs) with |log2Fold change| ≥ 1 and p-value < 0.05 were identified between M vs. X4, W vs. M, and W vs. X4, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses were performed to further annotate DEGs that were part of specific pathways, which revealed that out of 134 total pathways, MAPK signaling pathway, plant−pathogen interaction, and plant hormone signal transduction were highly enriched. Transcriptomic profiling analysis revealed that 1681 pathogen-related genes (PRGs), including 24 genes encoding WRKY transcription factors, potentially participate in the process of defense against Pestalotiopsis microspora infection in pecan. The correlation of WRKY TFs and PRGs was also performed to reveal the potential interaction networks among disease-resistance/pathogenesis-related genes and WRKY TFs. Expression profiling of nine genes annotated as TIFY, WRKY TF, and disease-resistance protein-related genes was performed using qRT-PCR, and the results were correlated with RNA-Seq data. This study provides valuable information on the molecular basis of pecan−Pestalotiopsis microspora interaction mechanisms and offers a repertoire of candidate genes related to pecan fruit response to FBS infection.
Topics: Carya; Pestalotiopsis; Plant Growth Regulators; Transcription Factors; Transcriptome
PubMed: 36232919
DOI: 10.3390/ijms231911621 -
Plant Disease Oct 2022Chinese figwort ( Hemsl.) is an important annual herb and its dried root tubers are used as a traditional Chinese herbal medicine. In May 2021, a disease with stem rot...
Chinese figwort ( Hemsl.) is an important annual herb and its dried root tubers are used as a traditional Chinese herbal medicine. In May 2021, a disease with stem rot symptoms on was observed at three randomly selected fields (~0.67 ha per field) in Nanchuan district (28.93°N, 107.27°E) of Chongqing, China. Disease incidence was estimated between 10% and 17% based on calculating the proportion of symptomatic plants. Initially, watery dark brown spots appeared on the epidermis of the stem. Then the spots expanded into spindle or strip shape, and the center of lesions were sunken, constricted and rotted finally (Figure 1A and Figure 1B). Leaves turned yellow and the plants wilted (Figure 1C). The infected parts of the stem broke easily and became brittle. The number of daughter buds used for reproduction was reduced by more than 24% and the production of root tubers decreased by more than 3%. Twelve stems with typical rot symptoms were sampled from the three fields for further investigation. Infested tissue fragments (4×4 mm) were surface sterilized with 75% ethanol for 30s and 2% sodium hypochlorite for 2 minutes in turn, finally, were rinsed 4 times with sterilized water. The disinfected tissue were air-dried and transferred onto potato dextrose agar (PDA) in the dark for 6 days at 25℃. The resulting fungal colonies were isolated by the single-spore isolation technique (Fang. 1998). Six different fungal colonies were isolated (X1-X6) and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The stem surfaces of 8 months old plants were sterilized with 75% ethanol for 30 s, rinsed three times with sterilized water, and stabbed with a sterilized needle. Conidial from the fungal colonies grown on PDA plate were harvested by filtration through five layers of sterilized absorbent gauze. Conidial concentration was then adjusted to 106 conidia per mL. 10 μL of conidial suspension was sprayed on stems injured with a sterile syringe. For each isolate, 6 plants were inoculated. Stems inoculated with sterilized water were used as a blank control. All plants were all put in a growth chamber at 28℃ with 75 to 80% relative humidity under a 12 h photoperiod for 15 days. The pathogenicity test was repeated once. After 13 days, the stems inoculated with X3 showed the same rot symptoms as we observed in the fields (Figure 1D) whereas the control stems remained symptomless (Figure 1E). The fungus re-isolated from the plants showing 100% symptoms had a similar morphology than X3 as described below. At the same time, the stems inoculated with X1, X2, X4, X5 and X6 showed no sign of rot. After culturing on PDA for 9 days under 25℃ in dark, isolate X3 grew all over the dish with white or pale pink pigmentation in the center (Figure 1F). Macroconidia were produced on synthetic low nutrient agar (SNA) plates, which showed sickle or spindle, 3 septate, straight to slightly curved with a foot-shaped basal cell, ranging from 17.595~44.88 × 2.04~3.315 μm (n=30). Microconidia were oval, elliptical or reniform, 0 to 1 septate, 3.06~12.75 ×1.785~2.805 μm (n=30) in size (Figure 1G). Phialides of conidiophores were cylindrical, short and monophialides or polyphialides (Figure 1H). Chlamydospores were found terminal or cluster with round or oblong (Figure 1I). These morphological characteristics described as (Skovgaard et al. 2003). For molecular identification, the ribosomal internal transcribed spacer (), translation elongation factor 1-alpha (), RNA polymerase II subunit 1 (), the largest subunit of RNA polymerase Ⅱ gene sequences () and the mitochondrial small subunit rDNA () genes were amplified with primers V9G /ITS4 (Hoog et al. 1998; White et al. 1990), EF1-668F /EF1-1251R (Alves et al. 2008), Fa/G2R (O'Donnell et al. 2010), 5f2/7cr (Liu et al. 1999; O'Donnell et al. 2010) and NMS1/NMS2 (Li et al. 1994). The sequences of isolate X3 were deposited in GenBank (MZ571935 (), MZ576201 (), MZ882396 (), MZ882397 () and MZ867716 ()). All sequences were revealed more than 99.8% sequence identity with reported sequences of (GenBank accession No: KY630717, JF740838, KU171680, KU171700 and MK439851). Based on the optimal nucleotide replacement model SYM of multi-gene series sequence matrix, the system development tree was constructed. Results showed the strain X3 and those of (Isolates numbers were NRRL 28387, MRC 2566, MRC 2564 and CZ3-5-6) were clustered into the same evolutionary branch with a post-mortem probability of 0.996 (Figure 2). According to the morphology, molecular identification and phylogenetic analysis based on the concatenated of and genes sequences, the isolated X3 was identified as . The ITS sequences of X1, X2, X4, X5 and X6 showed homology exceeding 97.1% to (MH931273), (MH858371), sp. (JX179237), (EF026129) and (EU552147), respectively, suggested the five strains to be these species possibly. GeneBank accession number of X1, X2, X4, X5 and X6 was OM074010, OM074011, OM074013, OM074015 and OM074018, respectively. To our best knowledge, this is the first report of infecting in China. Stem rot caused by is a severe threat to Chinese figwort cultivation, and identification of this pathogen is important for effective disease management and control.
PubMed: 36222729
DOI: 10.1094/PDIS-09-21-1896-PDN -
Plant Disease Oct 2022Machilus pauhoi Kaneh. is an excellent evergreen broad-leaved tree species widely grown in China for its ornamental and economic value (He et al. 2022). In September...
Machilus pauhoi Kaneh. is an excellent evergreen broad-leaved tree species widely grown in China for its ornamental and economic value (He et al. 2022). In September 2021, a leaf spot was observed on M. pauhoi plants on Guantian forest farm (27°06'15.6″N, 114°34'20.72″E) in ji' an city, Jiangxi province, China. The disease incidence was estimated to be above 20%. The symptoms began as brown irregular spots, then the spots gradually expand over time, with a gray-to-brown center and dark brown-to-black edges. Small infected tissues (3 to 5 mm2) were surface-sterilized in 70% ethanol for 30 s and 2% NaClO for 60 s, and rinsed three times with sterile water (Ju et al. 2021). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Colletotrichum ssp. were obtained (isolation frequency about 78%). Three representative single-spore isolates (PN-1, PN-4, and PN-9) were used for morphological studies and phylogenetic analyses. Colonies on the PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 11.4-16.8 ×4.1-5.5 µm (13.2 ± 1.0 × 4.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.2-8.8 × 4.1-6.2 µm (6.7 ± 0.2 × 5.1 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, ON176154 - ON176156; ACT, ON185554 - ON185556; GAPDH, ON185563 - ON185565; TUB2, ON185566 - ON185568; CHS-1, ON185560 - ON185562; CAL, ON185557 - ON185559). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed PN-1, PN-4, and PN-9 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on nine M. pauhoi plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto six leaves. Another three plants inoculated with ddH2O served as the control (Wan et al. 2022). All the inoculated leaves were covered with plastic bags to keep them moist for 2 days (relative humidity > 80%). All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 7 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Pestalotiopsis chamaeropis, Corynespora cassiicola and Arthrinium arundinis could infect M. pauhoi plants (Zhang et al. 2021), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on M. pauhoi. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.
PubMed: 36210334
DOI: 10.1094/PDIS-08-22-2005-PDN -
Plant Disease Oct 2022Thunb., an evergreen shrub, is popular for landscaping in China. In 2021, leaf spot was observed on (about 150 trees) leaves with 40 to 50% disease incidence in...
Thunb., an evergreen shrub, is popular for landscaping in China. In 2021, leaf spot was observed on (about 150 trees) leaves with 40 to 50% disease incidence in Wanzhou urban forest (30°45'N; 108°27'E) of Chongqing, the infected plants were between 5 and 6 years old. The symptoms started to occur from June to July and approximately 30 to 40% of the leaves exhibited leaf spot symptoms from August to September. Initial symptoms appeared as yellow spots of 1.2 to 4.9 mm in diameter, and then expanded to become large and irregular lesions, having white center surrounded by a brown halo. Under humid conditions, black dots appeared in the central part of the spots. In later stage, split and fall of the tissues occurred from the infected spot. To identify the causal agent, infected tissues from 20 samples (from 5 trees) were cut into small pieces (5 mm), surface-sterilized for 30 s in 75% ethanol and 3 minutes in 3% sodium hypochlorite, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml) and incubated at 25°C in dark conditions. Purified eight fungal colonies were white with undulating margins and light cream on the reverse side, measuring 85 mm diameter after 7 days, dark brown to black conidiomata were irregularly scattered and Conidia were observed in 20 days old colonies. Conidia were spindle-shaped, 4.5 to 6.8 × 15.2 to 23.5 μm (n=50), with 4 diaphragms, the three median cells were light to dark brown and the two end cells were colorless. 1 to 3 accessory filaments (5.2 to 22.5 μm long) protrude from theapical cell while a short stalk (3.5 to 5.5 μm long) was attached to the basal cell, these morphological features suggested that the isolates were most likely . sp. Eight colonies were confirmed to be identical based on morphological characteristics. For molecular identification, DNA was extracted from representative strains (YF-5, YF-13, YF-24). The internal transcribed spacer (ITS) region, β-tubulin (), the translation elongation factor-1 alpha gene (), genes were amplified using primers ITS5/ITS4, /, and /, respectively (White et al.1990; Glass & Donaldson 1995; O'Donnell & Cigelnik 1997; Carbone &, Kohn.1999). The sequences were deposited in NCBI GenBank YF-24, [ITS; ON204233: ; ON304156: ; ON400075]: YF-5, [ITS; OP379570: ; OP413495: ; OP413496]: YF-13, [ITS; OP379589: ; OP413494: ; OP413497]. Which revealed a 95% similarity to the NTUCC 18-067 [ITS; MT322086: ; MT321888: ; MT321987] ex-type sequences. Based on morphology and multilocus phylogenetic analysis, representative strains were identified as . For Koch's postulates, wiped the leaves of six healthy plants of (two-year-old) grown in pots with sterile water, 10 μL of spore suspension (10 spores/ mL) was brushed on five leaves per plant (three plants in total) with a sterile brush, and the other three plants were treated with sterile water instead of spore suspension as control, the plants were placed in a greenhouse at 28°C and 95±1% relative humidity. Seven days after inoculation, brown lesions appeared, similar to those observed in infected plants. Black dots surrounded by a brown halo reappear on the lesions after 12 days, whereas control plants remained healthy. culture was re-isolated from the infected leaves and identified using morphological characteristics and DNA sequence analysis. To our knowledge, can cause diseases on tea plants and has been found in Japan, Thailand and China, this is the first report of leaf spot infection of caused by in China. This disease is reducing the ornamental value of . Our results will contribute to the prevention and cure of leaf spot disease in .
PubMed: 36190304
DOI: 10.1094/PDIS-07-22-1722-PDN -
Molecular Plant-microbe Interactions :... Oct 2022
Topics: Ascomycota; Fragaria; Plant Diseases
PubMed: 36161793
DOI: 10.1094/MPMI-04-22-0077-A -
Frontiers in Microbiology 2022Post-harvest rot causes enormous economic loss to the global kiwifruit industry. Currently, there are no effective fungicides to combat the disease. It is unclear...
Post-harvest rot causes enormous economic loss to the global kiwifruit industry. Currently, there are no effective fungicides to combat the disease. It is unclear whether silver nanoparticles (AgNPs) are effective in controlling post-harvest rot and, if so, what the underlying antifungal mechanism is. Our results indicated that 75 ppm AgNPs effectively inhibited the mycelial growth and spore germination of four kiwifruit rot pathogens: , , , and . Additionally, AgNPs increased the permeability of mycelium's cell membrane, indicating the leakage of intracellular substance. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that AgNPs induced pathogen hypha shrinkage and distortion, as well as vacuolation in hypha cells, implying that AgNPs caused cellular and organelle structural degradation. The transcriptome sequencing of mycelium treated with AgNPs (24 h / 48 h) was performed on the Illumina Hiseq 4000 sequencing (RNA-Seq) platform. For the time points of 24 h and 48 h, AgNPs treatment resulted in 1,178 and 1,461 differentially expressed genes (DEGs) of , 517 and 91 DEGs of , 1,287 and 65 DEGs of , 239 and 55 DEGs of , respectively. The DEGs were found to be involved in "catalytic activity," "small molecule binding," "metal ion binding," "transporter activity," "cellular component organization," "protein metabolic process," "carbohydrate metabolic process," and "establishment of localization." Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that "carbohydrate metabolism," "amino acid metabolism," "energy metabolism," and "xenobiotics biodegradation and metabolism" of "metabolism processes" were the most highly enriched pathways for these DEGs in four pathogens, with "cellular processes" being particularly enriched for Furthermore, quantitative polymerase chain reactions (qPCRs) were used to validate the RNA-seq results. It was also confirmed that AgNPs could significantly reduce the symptoms of kiwifruit rot without leaving any Ag residue on the peel and flesh of kiwifruit. Our findings contributed to a better understanding of the antifungal effect and molecular mechanisms of AgNPs against pathogens causing kiwifruit post-harvest rot, as well as a new perspective on the application of this novel antifungal alternative to fruit disease control.
PubMed: 36118196
DOI: 10.3389/fmicb.2022.988633 -
Plant Disease Sep 2022Gardenia jasminoides Ellis, an important Chinese medicine, is cultivated on approximately 1,400 hectares in China. From August to October 2016, a severe disease...
Gardenia jasminoides Ellis, an important Chinese medicine, is cultivated on approximately 1,400 hectares in China. From August to October 2016, a severe disease affecting leaves, stems, and fruits of G. jasminoides, occurred in Cangnan (120°39'E, 27°48'N), Zhejiang province. Infected leaves or stems became shriveled, and in severe cases, young fruits presented red-brown or yellow necrotic lesions with numerous black spots. More than thirty diseased fruit and stem samples were collected, ten diseased fruits were surfacedisinfected (70% ethanol for 30 s, 1.5% sodium hypochlorite for 1 min) and kept at about 25℃ for 24 h with 80% humidity. The conidial suspension (105 conidia/ml) made of creamy drops secreted from the lesion black spots was spread onto PDA (Potato-Dextrose-Agar) and incubated at 25℃ in the dark for 7 d. Only one isolate (step01) was suspected to be the target pathogen, and other three isolates were Alternaria sp. The colony of step01 was white to grayish with an irregular edge on the front and a white to brown spiral grain on the back. Black pycnidia, produced after 20 d, were globose to subglobose, individual or overlapped, with an ostiole secreting a creamy conidial suspension. Alpha-conidia were aseptate, hyaline, oval to oblong with two oil balls, 7.4-15.9×2.4-4.5 µm (average 10.2×3.3 µm); beta-conidia were hyaline, aseptate, linetype, straight or slightly curled, 15.3-26.5×1.3-2.5 µm (average 20.8×1.6 µm). This isolate resembled Diaporthe sp. (Hansen and Barrett 1938). For species identification, DNA was extracted (Sangon Biotech Rapid Fungi Genomic DNA Isolation Kit - B518229),and the internal transcribed spacer region (ITS), elongation factor (EF1-α), β-Tubulin (TUB), and histone H3 (HIS) of step01 were amplified using the primer pairs ITS1/ITS4, EF1-728F/ EF1-986R, BT-2a /BT-2b and CYLH3F/CYLH3R, respectively (Udayanga et al 2014, Huang et al. 2015).These sequences were submitted to GenBank as KY797655 (ITS), MF158048 (EF1- α), MF158049 (TUB), and MF158050 (HIS). In comparison with the other sequence of Diaporthe sp. using MEGA7.0 (maximum likelihood, bootstrap replications=1,000), step01 showed 100% identity with D. gardeniae. Based on their morphology and molecular identification, step01 was identified as D. gardeniae (syn. Phomopsis gardeniae). Pathogenicity was tested on three one-year-old G. jasminoides plants by stem inoculation. Two or three stems per plant were inoculated by binding a mycelial plug (5 ×12 mm), covered by humid cotton and plastic film, to the tender stem. A total of two plants were treated. Plants were kept at about 25℃ for 4 weeks. Control plants were inoculated with PDA plugs. Leaf blight started from the apex, extended to the stalk, and the leaves finally fell off. Three months after inoculation, symptoms developed on the underlying leaves, the stem was withered with black spots, a pattern like that observed in the field. No symptoms appeared in the control leaves. Five identical colonies were re-isolated from symptomatic tissues and identified again as D. gardeniae, fulfilling the Koch's postulates. Several fungi are associated with canker, leaf spot, and fruit rot in Gardenia throughout China, including Pestalotiopsis sp. (Huang et al. 2006), Botryosphaeria dothidea (Dong et al. 2016), and Phoma sp. (Luo et al. 2016). To the best of our knowledge, this is the first report of D. gardenia infecting G. jasminoides Ellis in Zhejiang Province, China.
PubMed: 36109874
DOI: 10.1094/PDIS-03-22-0460-PDN