First report of leaf spot disease caused by Pseudopithomyces chartarum on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) cultivation has gradually expanded in China but continues to be limited by challenges such as leaf spot disease. Between September 2022 and September 2023, a leaf spot disease was observed on approximately 30% of 'Lanjingling' blue honeysuckles grown in a 2.66 ha field (a total of about 11,000 plants) in Jiamusi city (130.47°E, 46.16°N), Heilongjiang Province, China. Affected plants displayed brown necrotic lesions on their leaves that gradually expanded in area until the leaves fell off the plant entirely. Small, 3 to 4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Yan et al. 2022). Five pathogens (LD-232, LD-233, LD-234, LD-235, and LD-236) were isolated on PDA and displayed a conidia morphology consistent with Pseudopithomyces spp. (Perelló et al. 2017). The fungal colonies on PDA were villiform, white, and whorled and had sparse aerial mycelium on the surface with black conidiomata. The conidia were obpyriform and dark brown, had 0 to 3 transverse and 0 to 1 longitudinal septa, and measured 9.00 to 15.30 µm × 5.70 to 9.30 µm in size (n = 50). Genomic DNA was extracted from a representative isolate, LD-232, for molecular verification and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), LROR/LR7 (Carbone and Kohn 1999), and RPB2-5F2/RPB2-7CR (Liu et al. 1999) primers. Sequences of LD-232 ITS (OR835654), LSU (OR835652), and RPB2 (OR859769) revealed 99.8% (530/531 nt), 98.8% (639/647 nt), and 99.8% (1015/1017 nt) shared identity with Pseudopithomyces chartarum sequences (OP269600, OP237014, and MK434892), respectively (Wu et al. 2023). Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayes v. 3.2.7 to confirm the identity of the isolates (Ariyawansa et al. 2015). Phylogenetic trees cannot be constructed based on the genes' concatenated sequences because selective strains do not have complete rDNA-ITS, LSU, and RPB2 sequences. Therefore, based on the morphological characteristics and molecular phylogeny, LD-232 was identified as P. chartarum (Perelló et al. 2017; Wu et al. 2023). A pathogenicity test was performed with six healthy, two-year-old 'Lanjingling' blue honeysuckle plants. Three plants were inoculated by spraying the LD-232 conidial suspension (1 × 106 spores/ml) or clean water as an experimental control condition (Wu et al. 2023; Yan et al. 2023). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was replicated three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 days. The same pathogens were reisolated from infected leaves, displayed the same morphological and molecular traits, and were again identified as P. chartarum, confirming Koch's postulate. P. chartarum previously caused leaf spot disease on Tetrapanax papyrifer in China (Wu et al. 2023). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by P. chartarum in China. Identification of P. chartarum as a disease agent on blue honeysuckle will help guide future management of leaf diseases for this economically important small fruit tree.

First report of leaf spot disease caused by Neopestalotiopsis rosae on Lonicera caerulea L. in Heilongjiang Province, China.

Recently, interest in cultivating blue honeysuckle (Lonicera caerulea L.) for horticulture and medicinal uses has grown (Sharma and Lee 2021). Between September 2022 and September 2023, a leaf spot disease (Fig. S1) was observed on approximately 20% of 'Lanjingling' blue honeysuckles grown in a 0.18 ha field in Qiqihar city (123.43°E, 47.92°N), Heilongjiang Province, China. Infected plants displayed black leaf spots that expanded to cover the entire leaf. Small, 3 to 4 mm segments of infected tissue were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Ma et al. 2023). To induce sporulation, nine purified cultures (Fig. S2) with similar culture characteristics were finally obtained from ten infected plants and they displayed a conidia morphology consistent with Neopestalotiopsis spp., no other fungi were isolated, and the isolation frequency was 90%. Conidiomata (Fig. S3) were brown to black and distributed in concentric rings with an average size of 261.98 (60.30-451.80) µm (n = 50). The conidia (Fig. S3) were fusoid and had four septa, straight to slightly curved, with an average size of 23.48 (13.50-30.30) × 5.42 (4.50-9.30) µm(n = 50), while basal and apical cells were hyaline and the three middle cells were brown with darker septa. PCR amplification was performed with ITS1/ITS4 (White et al. 1990), EFl-728F/EF1-986R (Carbone and Kohn 1999), and Btub2Fd/Btub4Rd (Glass and Donaldson 1995) primers from the genomic DNA of the LD-330. Sequences of ITS (PP033584), TEF (PP048757), and TUB (PP048758) revealed 99 to 100% (499/500, 255/255, and 481/486) shared identity with Neopestalotiopsis rosae sequences (NR145243, KM199524, and KM199430) (Rebollar-Alviter et al. 2020). Therefore, based on morphological characteristics and molecular phylogeny, LD-330 was identified as N. rosae. Six two-year-old healthy plants of the 'Lanjingling' cultivar were selected for a pathogenicity test (Yan et al. 2023). The leaves were surface disinfected with 75% alcohol and then wiped with sterilized water three times. Three plants were inoculated with 10 ml of LD-330 conidial suspension (1 × 106 spores/ml) or with sterile water as an experimental control, respectively. All plants were in closed plastic bag, incubated in a greenhouse at 28 ℃ and 75% relative humidity (RH) under a 12-h light/dark cycle, and each experiment was performed three times (Rebollar-Alviter et al. 2020). Typical leaf spot symptoms were observed on inoculated leaves after 14 days (Fig. S4), whereas no symptoms were detected on water-treated leaves. The same pathogen was reisolated from infected leaves, displayed the same morphological and molecular traits, and was again identified as N. rosae, confirming Koch's postulate. Neopestalotiopsis rosae was previously reported on pecan (Gao et al. 2022), causing black leaf spot disease in China. To our knowledge, this is the first report of a blue honeysuckle leaf spot caused by N. rosae in China and specifically in the Heilongjiang province which has the largest blue honeysuckle cultivation area in the country. Future research should be directed toward developing comprehensive management measures.

First Report of Postharvest Fruit Rot Caused by Botrytis cinerea on Blue Honeysuckle (Lonicera caerulea L.) Fruit in China.

Blue honeysuckle (Lonicera caerulea L.) fruit is growing in popularity as a natural, functional 'super fruit', but its storage is challenged by pathogen infection. In June 2022, approximately 30% of 100 kg of blue honeysuckle fruits (cv. Lanjingling) obtained in Harbin, China (128.70°E, 44.87°N) showed postharvest fruit rot symptoms after 15 d of storage at 4°C, leading to whole fruit rotting with gray fungal growth (Fig.1 A). Small (1-2 mm) segments of infected tissue were obtained from 20 randomly selected fruits which were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Five purified cultures were obtained and their front colonies were dark brown (Fig.1 C) on the PDA plates after 5 d at 25°C (Alam et al. 2019; Riquelme-Toledo et al. 2020). The conidia (n = 50) were single-celled, hyaline, either ellipsoid or ovoid, and measured 7.5-15.0 µm (11.7 µm average) × 6.0-11.4 µm (8.3 µm average). The conidiophores (Fig.1 E) were branched at the apex bearing bunches of conidia resembling grape clusters (Ellis 1971). For molecular confirmation, genomic DNA was extracted from a representative isolate LDGS-3 using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer region (ITS, GenBank ON952502), heat shock protein (HSP60, GenBank OP039103), the second-largest subunit of RNA polymerase II (RPB2, GenBank OP186114) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH, GenBank OQ658508) genes were partially amplified with the respective primers ITS1/ITS4, HSP60f/HSP60r, RPB2f/RPB2r, and G3PDH-F/G3PDH-R (Staats et al. 2005; White et al. 1990). BLAST analysis revealed that the sequences of the four genes showed 100% homology with the MH782039, MH796663, MN448501 and MH796662 sequences for isolates of Botrytis cinerea. Based on morphology and molecular characteristics, the isolate LDGS-3 was identified as B. cinerea. For pathogenicity, twenty healthy blue honeysuckle fruits (cv. Lanjingling) were superficially sterilized with 75% ethanol and washed with distilled water. Ten inoculated blue honeysuckle fruits, which were injected with 10 µL conidial suspension of isolate LDGS-3 (106 spores/mL) displayed fruit rot symptoms (Fig.1 B) inside 9 cm Petri dishes after 10 d at 4°C, while no symptoms were detected on ten fruits inoculated with sterile distilled water (Alam et al. 2019). The same isolate that was reisolated from infected fruits with the same morphological and molecular traits was also identified as B. cinerea, confirming Koch's postulates. B. cinerea was previously reported in Henan Province, China in hawthorn (Zhang et al. 2018). To our knowledge, this is the first report of postharvest fruit rot caused by B. cinerea on blue honeysuckle fruit in China, which will aid future management of this emerging postharvest disease.

First report of Didymella glomerata causing leaf spot on Lonicera caerulea L. in Heilongjiang province, China.

China has the largest blue honeysuckle (Lonicera caerulea L.) cultivation area globally. In June 2022, leaf spots were observed on approximately 10% of blue honeysuckle (cv. 'Lanjingling') leaves in a 0.03-ha field in Harbin (127.66°E, 45.61°N), Heilongjiang Province, China. The leaves of the affected plants displayed chlorotic to tan dieback with a darker brown margin along the leaftip and leave margins. Cross-sectional segments of approximately 3 mm were cut from 50 typical infected plant leaves. Their surfaces were sterilized with 75% ethanol for 30 s followed by 3 min in 5% sodium hypochlorite (NaOCl), rinsed three times with sterile water, and transferred to 9-cm Petri dishes containing 15 ml of sterile PDA growth medium. Five purified cultures with similar culture characteristics were finally obtained and their colonies were dark brown on the PDA plates. The pycnidia were subglobular and deep black and measured avg. 215.48 (135.30-331.20) µm × avg. 170.28 (99.90-282.90) µm (n = 50) (Chen et al., 2015; Huang et al., 2018). Conidia were single-celled, hyaline, and ellipsoidal and measured avg. 6.22 (5.40-7.20) µm × avg. 3.42 (2.70-3.90) µm (n = 50). For molecular verification, genomic DNA was extracted from a representative isolate, LD-75. The internal transcribed spacer region (ITS), the second-largest subunit of RNA polymerase II (rpb2), the partial 28S large subunit rDNA (LSU), beta-tubulin (TUB), and actin (ACT) genes were amplified with the primers ITS1/ITS4, RPB2f/RPB2r, LROR/LR7, TUB2Fd/TUB4Rd, and ACT512f/ACT783R, respectively (White et al. 1990; Carbone and Kohn, 1999; Staats et al., 2005; de Gruyter et al., 2009; Chen et al., 2015). BLAST results indicated that the genes of LD-75 (GenBank OP218870, OP264863, OQ561448, OQ597233, and OQ597232) shared 99%-100% identity with those of Didymella glomerata (OK485138, GU371781, EU754185, MZ073910, and MW963190, respectively). Therefore, based on morphological characteristics and molecular phylogeny, LD-75 was identified as D. glomerata. Six two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. The leaves were surface disinfested with 75% ethanol and then wiped with sterilized water three times. All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle. Whole plants sprayed with conidial suspension of isolate LD-75 (106 spores/mL) (n = 3) displayed leaf spot symptoms after 14 d, while no symptoms were detected on whole plants sprayed with sterile water (n = 3). The same isolate, reisolated from infected leaves and with the same morphological and molecular traits, was also identified as D. glomerata, confirming Koch's postulate. The fungus was previously reported in Cornus officinalis in Nanyang City, China (Huang et al., 2018). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by D. glomerata in China. Reducing blue honeysuckle production losses caused by leaf spots is crucial for growers, and we hope that researchers will develop efficient control strategies for managing this emerging plant disease.

First report of leaf spot disease caused by Alternaria tenuissima on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) is a perennial plant of the Caprifoliaceae family and Lonicera genus, the largest genus in the plant kingdom. Between September 2021 and September 2022, a leaf spot disease was observed on ~20% of blue honeysuckles of the 'Lanjingling' cultivar grown in a 3.33 ha field at the Xiangyang base (126.96°E, 45.77°N) of the Northeast Agricultural University, Harbin (Heilongjiang Province, China). Leaf spots first presented black mildew centers, gradually covering large areas of the leaf until it eventually fell off. Small 3-4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol and 5% sodium hypochlorite, rinsed in sterile distilled water, and transferred to 9 cm Petri dishes containing potato dextrose agar (PDA) after drying. Finally, two isolated pathogens were obtained through single spore culture on PDA; they appeared as gray-black colonies and were named LD-12 and LD-121. The observed LD-12 and LD-121 conidia displayed a morphology consistent with Alternaria spp. They were obpyriform and dark brown, with 0-6 transverse and 0-3 longitudinal septa, measuring 6.00-17.70 µm × 9.30-42.30 µm and 5.70-20.70 µm × 8.40-47.70 µm for LD-12 and LD-121, respectively (n = 50). Genomic DNA was extracted from the two isolates for molecular verification, and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005) primers. Sequences of LD-12 ITS (OQ607743), GPD (OQ623200), TEF (OQ623201), RPB2 (OQ658509), and ALT (OQ623199) revealed 99-100% of identity with Alternaria tenuissima sequences (KC584567, MK451973, LT707524, MK391051, and ON357632). Sequences of LD-121 ITS (OQ629881), GPD (OQ850078), TEF (OQ850075), RPB2 (OQ850076), and ALT (OQ850077) revealed 99-100% identity with A. alternata sequences (MN826219, ON055384, KY094927, MK637444, and OM849255). Nine two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. Three plants were inoculated with either the LD-12 or LD-121 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control condition (Mirzwa-Mróz et al., 2018; Liu et al., 2021). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was performed three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 d. The same pathogens reisolated from infected leaves displayed the same morphological and molecular traits. They were again identified as A. tenuissima and A. alternata, confirming Koch's postulate. A. tenuissima and A. alternata were previously reported on Orychophragmus violaceus (Liu et al., 2021) and L. caerulea (Yan et al., 2022) in China. This study is the first report of a blue honeysuckle leaf spot caused by A. tenuissima in China. In the future, effective biological and chemical control should be used to prevent blue honeysuckle leaf spots in China.

A Novel Estrone Degradation Gene Cluster and Catabolic Mechanism in Microbacterium oxydans ML-6.

Global-scale estrone (E1) contamination of soil and aquatic environments results from the widespread use of animal manure as fertilizer, threatening both human health and environmental security. A detailed understanding of the degradation of E1 by microorganisms and the associated catabolic mechanism remains a key challenge for the bioremediation of E1-contaminated soil. Here, Microbacterium oxydans ML-6, isolated from estrogen-contaminated soil, was shown to efficiently degrade E1. A complete catabolic pathway for E1 was proposed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR). In particular, a novel gene cluster (moc) associated with E1 catabolism was predicted. The combination of heterologous expression, gene knockout, and complementation experiments demonstrated that the 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) encoded by the mocA gene was responsible for the initial hydroxylation of E1. Furthermore, to demonstrate the detoxification of E1 by strain ML-6, phytotoxicity tests were performed. Overall, our findings provide new insight into the molecular mechanism underlying the diversity of E1 catabolism in microorganisms and suggest that M. oxydans ML-6 and its enzymes have potential applications in E1 bioremediation to reduce or eliminate E1-related environmental pollution. IMPORTANCE Steroidal estrogens (SEs) are mainly produced by animals, while bacteria are major consumers of SEs in the biosphere. However, the understanding of the gene clusters that participate in E1 degradation is still limited, and the enzymes involved in the biodegradation of E1 have not been well characterized. The present study reports that M. oxydans ML-6 has effective SE degradation capacity, which facilitates the development of strain ML-6 as a broad-spectrum biocatalyst for the production of certain desired compounds. A novel gene cluster (moc) associated with E1 catabolism was predicted. The 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) identified in the moc cluster was found to be necessary and specific for the initial hydroxylation of E1 to generate 4-OHE1, providing new insight into the biological role of flavoprotein monooxygenase.

Biological saccharification coupled with anaerobic digestion using corn straw for sustainable methane production.

In this study, accumulated fermentable sugars from biosaccharified corn straw were used to generate methane through anaerobic digestion (AD). The results showed that reducing sugars from biosaccharification expanded corn straw (BECS) treated with Clostridium thermocellum XF811 accumulated with yields of 94.9 mg/g. The BECS used for AD was converted into a high methane yield (7436 mL), which was 49.3 % higher than that of expanded corn straw (ECS). High-throughput microbial analysis suggested that Methanoculleus and Methanobacterium greatly contributed to the high methane yield. Industrial experiments demonstrated that the methane production from BECS by AD was 72,955 m3, which increased by 13.2 % compared to that from ECS. Biosaccharification pretreatment accelerated ECS destruction and accumulated sugars, thereby increasing methane yields. This study provides a strategy for producing clean energy from lignocellulose biomass.

Potential and mechanism for bioremediation of papermaking black liquor by a psychrotrophic lignin-degrading bacterium, Arthrobacter sp. C2.

To meet the challenge of bioremediation of black liquor in pulp and paper mills at low temperatures, a psychrotrophic lignin-degrading bacterium was employed in black liquor treatment for the first time. In this study, Arthrobacter sp. C2 exhibited excellent cold adaptability and lignin degradation ability, with a lignin degradation rate of 65.5% and a mineralization rate of 43.9% for 3 g/L lignin at 15 °C. Bioinformatics analysis and multiple experiments confirmed that cold shock protein 1 (Csp1) was the dominant cold regulator of strain C2, and dye-decolorizing peroxidase (DyP) played a crucial role in lignin degradation. Moreover, structural equation modeling (SEM), mRNA monitoring, and phenotypic variation analysis demonstrated that Csp1 not only mediated cold adaptation but also modulated DyP activity by controlling dyp gene expression, thus driving lignin depolymerization for strain C2 at low temperatures. Furthermore, 96.4% of color, 64.2% of chemical oxygen demand (COD), and 100% of nitrate nitrogen (NO3--N) were removed from papermaking black liquor by strain C2 within 15 days at 15 °C. This study provides insights into the association between the cold regulator and catalytic enzyme of psychrotrophic bacteria and offers a feasible alternative strategy for the bioremediation of papermaking black liquor in cold regions.

Efficient conversion of hemicellulose into 2, 3-butanediol by engineered psychrotrophic Raoultella terrigena: mechanism and efficiency.

Low-temperature biorefineries inhibit the multiplication of undesired microorganisms, improve product purity and reduce economic costs. Herein, to improve the 2,3-butanediol (2,3-BD) bioconversion efficiency from hemicellulose, a psychrotrophic hemicellulose-degrading strain Raoultella terrigena HC6 with high ß-xylosidase activity 1520 U/mL was isolated and genetically modified. Xylan (hemicellulose replacement) was depolymerized into xylooligosaccharides (XOS) and xylose by HC6, which were further converted into 2,3-BD. Transcriptomic analysis revealed that ß-xylosidase gene (xynB) and xylose isomerase gene (xylA), which are beneficial for increasing the carbon flux from xylan to 2,3-BD, were significantly upregulated 56.9-fold and 234-fold, respectively. A recombinant strain was constructed by overexpressing xynB in HC6, which obtained 0.389 g/g yield of 2,3-BD from hemicellulose extracted from corn straw at 15 °C. This study proposed a promised strategy for the bioconversion of agricultural waste into 2,3-BD at low temperatures and provides a basis for future efforts in the achievement of carbon neutrality.

First report of leaf spot disease caused by Alternaria alternata on Lonicera caerulea L. in China.

Relatively few studies investigating plant diseases in blue honeysuckle (Lonicera caerulea L.) have been conducted in China. In September 2021, a leaf spot disease was observed on approximately 10% of blue honeysuckle 'Lanjingling' leaves in a 0.02 ha field plantation in Tiekuang Street (124.36°E, 40.12°N) in Dandong City, Liaoning Province, China. The main symptom consisted of leaf spots with black mildew centers typically surrounded by yellow halos. Small pieces (3-4 mm) of the infected leaves were plated onto potato dextrose agar (PDA) medium as described by Wang et al. (2020) and six purified cultures were obtained through single spore culture on PDA. The observed conidia, consistent with the morphology of Alternaria alternata, were obpyriform and dark brown, measuring 5.8 to 15.3 µm × 7.9 to 42.5 µm, with 1-6 transverse septa and 0-3 longitudinal septa (n = 50) (Simmons 2007). For molecular verification, genomic DNA was extracted from a representative isolate LD-8. The ITS (GenBank OL454815), GPD (GenBank OL601993), TEF (GenBank OL538256), RPB2 (GenBank OL601966), and Alt (GenBank OL538257) genes were partially amplified with the respective primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EFI-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005). BLAST analysis revealed that these genes shared 99%-100% identity with OK345332, MK451977, MN756011, KU933459, and MN655781, respectively. A greenhouse experiment was conducted using six, healthy two-year-old blue honeysuckle 'Lanjingling' plants to observe disease development (Mirzwa-Mróz et al. 2018). After 10 d, we noted typical leaf spot symptoms on inoculated leaves sprayed with a conidial suspension (106 spores/mL) while no symptoms were detected on uninoculated leaves. The same isolate, reisolated from infected leaves with the same morphological and molecular traits, was also identified as A. alternata, confirming Koch's postulates. The fungus was previously reported in cockscomb plants in Heilongjiang Province, China (Wang et al. 2020). To our knowledge, this is the first report of leaf spot disease caused by A. alternata in blue honeysuckle grown in China. This study will provide a basis for future development of effective protection strategies against blue honeysuckle leaf spot in China.