G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review.

Sclerotinia sclerotiorum is an economically damaging fungal pathogen that causes Sclerotinia stem rot in legumes, producing enormous yield losses. This pathogen is difficult to control due to its wide host spectrum and ability to produce sclerotia, which are resistant bodies that can remain active for long periods under harsh environmental conditions. Here, the biocontrol methods for the management of S. sclerotiorum in legumes are reviewed. Bacillus strains, which synthesized lipopeptides and volatile organic compounds, showed high efficacies in soybean plants, whereas the highest efficacies for the control of the pathogen in alfalfa and common bean were observed when using Coniothyrium minitans and Streptomyces spp., respectively. The biocontrol efficacies in fields were under 65%, highlighting the lack of strategies to achieve a complete control. Overall, although most studies involved extensive screenings using different biocontrol agent concentrations and application conditions, there is a lack of knowledge regarding the specific antifungal mechanisms, which limits the optimization of the reported methods.

Occurrence and management of the emerging pathogen Epicoccum sorghinum.

Epicoccum sorghinum is a notorious fungal pathogen that causes leaf spot symptoms on a wide range of plants, leading to devastating losses in crop production and quality. Here, all reports regarding the occurrence and management of E. sorghinum are covered for the first time. E. sorghinum has been detected in tropical and subtropical climate areas during the rainy season, mainly from March to August, since 2016. Although E. sorghinum shows broad host spectrum, the disease incidence is especially notorious in cereal crops and ornamental plants, suggesting that these plants are especially susceptible. Control methods based on synthetic fungicides, plant extracts, and microbial biocontrol agents have been reported. However, most agents were applied using only in vitro conditions, restricting the information about their actual applicability in field conditions. Additionally, E. sorghinum can colonize cereal grains and synthesize the carcinogenic mycotoxin tenuazonic acid, posing an enormous hazard for human health. Furthermore, although E. sorghinum is an emerging pathogen that is currently causing yield penalties in important crops, there is lack of information about its pathogenic mechanisms and virulence factors, and there is currently no commercial antifungal agent to manage E. sorghinum. Collectively, it is imperative to conduct in vivo studies to determine the efficacy of antifungal agents and the most effective methods of application in order to develop suitable management strategies against E. sorghinum.

First Report of Penicillium oxalicum Causing Leaf Blight on 'Hongyang' Kiwifruit in China.

In September 2022, leaf blight symptoms (Fig. 1) were detected on six-year-old kiwi trees (Actinidia chinensis cv. 'Hongyang') in Xuzhou municipality (117.29º E, 34.23º N), Jiangsu Province. Early-stage disease symptoms included light brown necrotic lesions of irregular shape ranging in length from 0.2 to 2.4 cm, which turned into leaf blight after approximately 2 weeks. Those symptoms were similar to those previously reported during a Pestalotiopsis sp. infection on kiwi trees in Turkey (Karakaya 2001). Approximately 20% of the leaves from 300 trees examined in one kiwi orchard, 3000 m2 in size, showed the disease symptoms. Ten leading edges of symptomatic leaves were sterilized with 2% sodium hypochlorite for 1 min, rinsed twice with sterile ddH2O and cultured at 26ºC for 3 days on PDA medium containing 50 µg/ml chloramphenicol. The fungal colonies were collected, and the single spore isolation method was used to obtain four isolates. The obtained isolates showed white aerial mycelia that turned greyish after 2 days of cultivation on PDA medium at 26ºC. ITS (OR054113, OR054153-OR054155), TUB2 (OR060951-OR060953, OR249978), and CMD (OR255947-OR255950) genes were amplified using the ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively (Visagie et al. 2014a). The obtained ITS, TUB2, and CMD sequences shared 99.81%-100%, 96.72%-96.96%, and 90.17%-92.58% homology compared to the ex-type strain P. oxalicum CBS 219.30 (MH855125, KF296462, and KF296367), while the obtained ITS and TUB2 sequences showed 99.62%-99.81%, and 96.46%-96.72% identity compared to the representative strain P. oxalicum DTO 179B9 (KJ775647 and KJ775140) (Visagie et al. 2014b). The sequences obtained also showed high homology compared to P. oxalicum HP7-1 (ITS: 99.81%-100% homology; TUB2: 98.98%-99.38% homology; CMD: 94.71%-95.10% homology) (Li et al. 2022). A molecular phylogenetic tree was constructed using MEGA X with representative Penicillium strains retrieved from GenBank (Fig. 2). Microscope observations revealed the presence of curved septate hyphae. Conidia were colorless, unicellular, and ellipsoidal (5-8 µm in length; > 2000 observations), whereas conidiophores were mainly monoverticillate (approximately 20% of the conidiophores were biverticillate) (50-70 µm in length; 43 observations) and contained cylindrical phialides (13-15 µm in length). These findings are consistent with P. oxalicum morphology (Wu et al. 2022; Zheng et al. 2023). The pathogenicity of the four isolates was screened using healthy non-detached 'Hongyang' kiwi leaves. Fifteen leaves from five different two-month-old trees were used for each isolate, with three repetitions. For inoculation, a 10 mL solution containing 1 × 106 spores/mL was sprayed on the leaves. Sterilized water was used in the control experiment, which was carried out using fifteen leaves from five different two-month-old trees, with three repetitions. Inoculated trees were stored at 26ºC and 60% relative humidity for 2 days. All the infected leaves had necrotic lesions and leaf blight symptoms comparable to those found in the field, but the control leaves had no lesions. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphology analysis, fulfilling Koch's postulates. P. oxalicum is a common cause of blue mould in postharvest fruits (Tang et al. 2020). P. oxalicum has been recently reported as the causal agent of leaf spot in pineapple (Wu et al. 2022; Zheng et al. 2023), and leaf blight on maize (Han et al. 2023). Although Alternaria sp., Glomerella cingulate, Pestalotiopsis sp., Phomopsis sp., and Phoma sp. were previously isolated from kiwi leaves with blight symptoms (Kim et al. 2017), this is the first report of P. oxalicum causing leaf blight on kiwi trees worldwide. P. oxalicum is a well-known source of mycotoxins, such as secalonic acid (Otero et al. 2020), indicating that its presence in kiwifruit orchards may pose a significant risk to human health. The discovery of this hazardous pathogen in kiwi trees must drive the development of management strategies. Kiwifruit is an important dietary source of vitamins, fiber, folate, and potassium, and China is the major producer of kiwifruit, with more than 1.2 million metric tons harvested in 2021. This report will help to generate a better understanding of the pathogens affecting kiwifruit orchards in China.

First Report of Penicillium oxalicum Causing Leaf Blight on Maize in China.

In August 2022, two-month-old maize plants (Zea mays cv. 'Zihei'; "Chinese purple corn") exhibited irregular lesions on leaves and leaf blight symptoms (Figure 1). Although the lesions were yellow at the early infection stages, they turned brown during the pathogen advancement and culminated in leaf blight. Nearly 60% of plants from a non-commercial maize field (0.2 ha) in south-eastern Jiangsu (Nantong municipality, China; 120.54º E, 31.58º N) exhibited brown lesions, and about 4% of the diseased plants showed advanced leaf blight symptoms. The disease resulted in approximately a 9% yield loss compared to previous years when no disease symptoms were observed. Thirty small leaf pieces, approximately 0.3 cm2 in size and showing disease symptoms, were surface sterilized in 1.5% NaOCl for 1 min and washed twice with sterile ddH2O. The pathogen was cultured on PDA medium in the dark at 25 ºC, with grayish colonies observed after 5 days. Morphological analysis showed the presence of round/oval conidia (8.81 ± 0.50 µm diameter; n = 86) and branched conidiophores, which was consistent with the morphology of Penicillium spp. (Visagie et al. 2014). Nine representative isolates were obtained from different leaf pieces via single spore isolation, and the internal transcribed spacer (ITS), ß-tubulin (TUB2) and calmodulin (CMD) genes were amplified using ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively. The obtained ITS (OP954496-OP954497 and OP942428-OP942434), TUB2 (OP966781-OP966784 and OQ025045-OQ025049) and CMD (OQ078664-OQ078672) sequences were submitted in GenBank. Two isolates belonged to the P. citrinum species, while seven of the isolates belonged to the P. oxalicum species. A blast search revealed that the obtained P. citrinum ITS and CMD sequences had 99.39% and 100% homology to the ex-type strain P. citrinum NRRL 1841; GenBank numbers: AF033422 and GU944638 (Peterson & Horn 2009). Additionally, the obtained P. oxalicum ITS and CMD sequences had 99.82-100% and 94.64-95.49% homology to the ex-type strain P. oxalicum NRRL 787; GenBank numbers: AF033438 and KF296367 (Visagie et al. 2015). A molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen (Figure 2). To confirm pathogenicity, 3-week-old healthy 'Zihei' plants were used. The leaves were sprayed with aqueous solutions (sterilized ddH2O) that contained 1 × 106 spores/mL of each isolate. For the control experiment, sterilized ddH2O was used. After 5 days in a growth chamber at 25 ºC and 70% relative humidity, yellow lesions were observed. The number of lesions was higher when inoculating with P. oxalicum than when inoculating with P. citrinum. This result, together with the higher occurrence of P. oxalicum isolates, suggests that P. oxalicum is the main species causing the disease symptoms. The pathogen was recovered from the infected plants, and its identity was confirmed by ITS sequencing and morphological analysis. As far as we know, this is the first report of P. citrinum and P. oxalicum causing maize leaf blight worldwide. These species have previously been associated with maize kernels, as a source of mycotoxins posing relevant hazards to human health (Keller et al. 2013; Yang et al. 2020). P. citrinum was recently identified as the causal agent of green mold on Dictyophora rubrovalvata in China (Qin et al. 2022), while P. oxalicum was reported to cause citrus rot, pineapple leaf spot, and blue mold on Gastrodia elata, Astralagus membranaceus and muskmelon (Tang et al. 2020; Wu et al. 2022; Zheng et al. 2022). China is one of the world's largest producers of maize, harvesting more than 171 million tons in 2021. This report will help to better understand the pathogens that affect China's maize production.

First Report of Fusarium luffae Causing Leaf Blight on Loquat in Eastern China.

During May-June 2021 and 2022, leaf blight symptoms were observed on loquat leaves (Eriobotrya japonica cv. 'Mogi') in Jiangsu Province (Xuzhou municipality, 117.17° E, 34.13° N) in China. Approximately 10% of the leaves on the two hundred trees studied in a six-year-old loquat orchard exhibited round lesions that changed from light yellow to reddish-brown in 8-10 days. Approximately 3% of the infected leaves exhibited numerous lesions that coalesced, leading to expansive blighted areas. Twenty-five samples of symptomatic tissue, approximately 0.2 cm2 in size, were collected in May 2022 from five different trees (five samples per tree), sterilized in 2% NaOCl for 1 min, washed twice with sterilized ddH2O, and incubated at 26°C for 5 days on PDA medium containing 50 µg/mL chloramphenicol. Six isolates were obtained via single spore isolation. ITS (OQ954852-OQ954857), TUB2 (OQ968488-OQ968493), EF1-α (OQ971890-OQ971895), RPB1 (OQ971896-OQ971901), and RPB2 (OR037266-OR037271) genes were amplified using the ITS1/ITS4, T1/T22, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and fRPB2-7CF/fRPB2-11aR primers, respectively (O'Donnell et al. 2010). The species was identified using the Fusarioid ID database (Crous et al. 2021), revealing that all obtained isolates showed high homology to representative F. luffae strains. Upon combining the ITS, TUB2, EF1-α, RPB1, and RPB2 sequences, the isolates showed 99.42%-97.85% and 99.59%-98.10% identity to F. luffae CGMCC 3.19497 (ex-type strain) and NRRL 32522, respectively. A molecular phylogenetic tree was constructed using MEGA X, with a selection of representative Fusarium strains. Microscope observations showed septate mycelium, microconidia (6.86 ± 0.91 µm length, 1.67 ± 0.24 µm width, containing 1 septum; number of observations = 21), fusiform macroconidia (15.88 ± 1.43 µm length, 1.66 ± 0.24 µm width, containing 1 septum; number of observations = 45), and linear chlamydospores (79.36 ± 28.36 µm length, 12.03 ± 3.37 µm width; number of observations = 152). These observations are consistent with the morphology of F. luffae (Wang et al. 2019). All isolates exhibited identical morphological characteristics. All isolates were evaluated for pathogenicity in vivo using healthy non-detached loquat leaves. A total of 15 leaves from 5 different three-month-old 'Mogi' loquat trees were used for each isolate. Experiments were performed three times. A suspension of 1 × 106 spores/mL obtained from a seven-day-old colony (10 mL per 15 leaves), was sprayed on non-wounded leaves for inoculation. Sterilized ddH2O was used in the control experiment. Inoculated trees were stored at 26°C and 70% relative humidity for four days. Leaf blight symptoms were observed in all inoculated leaves, and the symptoms were observed in all repeated trials. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphological analysis, fulfilling Koch's postulates. In recent years, F. luffae has been reported to cause fruit rot on muskmelon, flower rot on kiwifruit, soybean pod rot, and leaf spot on cherry in China (Yu et al. 2022; Zhang et al. 2022; Zhao et al. 2022; Zhou et al. 2022), demonstrating the host promiscuity of this pathogen. Although F. solani has been identified as the causal agent of root rot and fruit rot on loquat (Abbas et al. 2017; Wu et al. 2021), this is the first report of F. luffae causing leaf blight on loquat worldwide. This report will help to understand the pathogens affecting loquat orchards in China.

Application of chitosan nanoparticles in quality and preservation of postharvest fruits and vegetables: A review.

Chitosan is an interesting alternative material for packaging development due to its biodegradability. However, its poor mechanical properties and low permeability limit its actual applications. Chitosan nanoparticles (CHNPs) have emerged as a suitable solution to overcome these intrinsic limitations. In this review, all studies regarding the use of CHNPs to extend the shelf life and improve the quality of postharvest products are covered. The characteristics of CHNPs and their combinations with essential oils and metals, along with their effects on postharvest products, are compared and discussed throughout the manuscript. CHNPs enhanced postharvest antioxidant capacity, extended shelf life, increased nutritional quality, and promoted tolerance to chilling stress. Additionally, the CHNPs reduced the incidence of postharvest phytopathogens. In most instances, smaller CHNPs (<150 nm) conferred higher benefits than larger ones (>150 nm). This was likely a result of the greater plant tissue penetrability and surface area of the smaller CHNPs. The CHNPs were either applied after preparing an emulsion or incorporated into a film, with the latter often exhibiting greater antioxidant and antimicrobial activities. CHNPs were used to encapsulate essential oils, which could be released over time and may enhance the antioxidant and antimicrobial properties of the CHNPs. Even though most applications were performed after harvest, preharvest application had longer lasting effects.

First Report of Fusarium acuminatum Causing Leaf Blight on Garlic in China.

In June 2021, leaf blight symptoms were detected on garlic plants (Allium sativum) in southeastern Jiangsu (Nantong municipality; 120.61° E, 33.25° N) in China. Two-month-old garlic plants exhibited leaf tip die back and light brown lesions in new and old leaves (Figure 1). The symptoms were observed in 40% of the plants in a 60-square-meters commercial field surrounded by rice fields, and were similar to those reported for Botrytis porri, Septoria allii and Stemphylium eturmiunum causing leaf blight on garlic (Dumin et al. 2021; Park et al. 2013; Zhang et al. 2009). Six samples of symptomatic tissue collected in Nantong municipality, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 15 min and washed twice with sterile ddH2O. The pathogen was isolated from all collected samples on PDA medium, containing 50 µg/mL chloramphenicol, at 26°C. Pink colonies with orange pigmentation were observed after 7 days. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes were amplified using ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5 and fRPB2-7CF/fRPB2-11aR primers, respectively. A total of 17 isolates were obtained, with nine of the isolates sharing the same sequences (strain NJC21), six of the isolates sharing the same sequences (strain NJC22), and the other two isolates showing different sequences (strains NJC23 and NJC24). The obtained sequences were submitted in GenBank under accession numbers OL655398-OL655401 (ITS), and OL741712-OL741723 (EF1-α, RPB1, RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from F. acuminatum IBE000006 (EF531232), the EF1-α sequences shared 99% homology to the EF1-α gene from F. acuminatum F1514 (LC469785), the RPB1 sequences shared >99% homology to the RPB1 gene from F. acuminatum JW 289003 (MZ921675), and the RPB2 sequences shared 100% homology to the RPB2 gene from F. acuminatum NL19-077002 (MZ921813) or 100% homology to the RPB2 gene from F. acuminatum MD1 (MW164629). A phylogenetic tree was constructed using MEGA7 with related Fusarium strains (Figure 2). Microscope observations after incubation in potato-sucrose-agar (PSA) medium showed the presence of oval microconidia, fusiform macroconidia, septate mycelium and chlamydospores, and agree with the morphology of F. acuminatum (Marek et al. 2013). The pathogenicity was screened with two-week-old wounded and non-wounded garlic plants using a 1 × 106 spores/mL solution (20 µL). Sterile ddH2O was used in the control experiment. The inoculated plants were incubated at 26°C and 60% relative humidity for 3 days, detecting similar lesions compared to those observed in the field. The pathogen was recovered from 5 different lesions, from different plants, and its identity was confirmed by sequence analysis. Recently, F. acuminatum was reported to cause garlic bulb rot in Serbia (Ignjatov et al. 2017). Although F. acuminatum is well known as a causal agent of root rot (Li et al. 2021; Tang et al. 2021), F. acuminatum has also been found causing leaf blight on onion (Parkunan et al. 2013) and muskmelon (Yu et al. 2021). This is the first report of F. acuminatum causing leaf blight on garlic, demonstrating the host and tissue promiscuity of this pathogen. China is the largest producer of garlic in the world with nearly 20 million tons harvested in 2020. This report will help to better understand the pathogens that are affecting garlic production in China.

First Report of Epicoccum sorghinum Causing Leaf Sheath and Leaf Spot on Maize in China.

In November 2020, leaf sheath on maize (Zea mays) was detected in southeastern Jiangsu (Nantong municipality; 120.54° E, 31.58° N) in China. Physiologically mature plants, 13 weeks of cultivation (at the harvest stage), exhibited red-brown lesions in stem and leaves, and dried-up stem (Figure 1). The symptoms were observed on approximately 95% of the maize plants in a 0.8 ha maize field surrounded by old sorghum fields and the crop yield was decreased by 70-85% with respect previous years, when no disease symptoms were detected. Small pieces, approximately 0.3 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated (one isolate was obtained) and cultured on PDA medium, containing chloramphenicol (50 µg/mL), under darkness at 26 ºC for 3 days. Amplification of internal transcribed spacer (ITS), large subunit (LSU), actin (ACT) and ß-tubulin (TUB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R and T1/Bt2b primers, respectively (Ma et al. 2021). Sequences were submitted to GenBank under accession numbers MW800180 (ITS), MW800361 (LSU), MW845677 (ACT) and MW892439 (TUB2). Blast search revealed that the ITS sequence had 100% (492/492 bp) homology with E. sorghinum LY-D-1-1, MT604999, LSU had 98% (1075/1091 bp) homology with E. sorghinum GZDS2018BXT010, MK516207, ACT had 96% (214/222 bp) homology with E. sorghinum M3, MK044832, and TUB2 had 99% (498/499 bp) homology with E. sorghinum BJ-F1, MF987525. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-1774.9882) is shown in Figure 2. Bipolaris, Curvularia and Fusarium spp. found causing leaf spot on maize were included in the phylogenetic tree (Liu et al. 2021; Reyes Gaige et al. 2020; Chang et al. 2016). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem and leaves of 2-week old maize plants. A solution containing 1 × 108 spores/mL (20 µL) was injected in the wound, whereas sterilized ddH2O was used in the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity for 3 days, observing fast-growing necrotic lesions in both stem and leaves. The pathogen was recovered from the infected plants and its identity was confirmed by morphological and sequence analyses. Microscope observations indicated the presence of chlamydospores, oval conidia (3 × 5 µm) and round pycnidia (60-100 µm diameter), and agree with those previously reported for the morphology of E. sorghinum (Bao et al. 2019). During last 2 years, E. sorghinum was reported to cause leaf spot on a number of relevant agricultural crops in China, including taro, Brassica parachinensis, tea, rice and wheat (Du et al. 2020; Li et al. 2020; Liu et al. 2020a, 2020b), confirming the expansion and host promiscuity of this pathogen. The pathogen was also reported to cause leaf spot on maize in Brazil in 2004 (Do Amaral et al. 2004); however, this is the first report of E. sorghinum causing leaf sheath and leaf spot on maize in China. Maize an important agricultural crop in China with more than 168 million tons produced in 2019. The observed yield loss and disease incidence of the isolated strain suggest that E. sorghinum may be a threat to maize production in China.

First Report of Colletotrichum brevisporum Causing Soybean Anthracnose in China.

In March 2020, widespread anthracnose was observed on soybean (Glycine max) in southeastern Jiangsu (Nantong municipality; 120.53° E, 31.58° N) in China. Plants exhibited irregular brown necrotic lesions in stem and leaves, and pronounced wilting. The symptoms were detected in one soybean field, 0.42 ha, surrounded by healthy wheat fields. Approximately 65% of the soybean plants showed the disease symptoms, and crop yield was reduced by 28-35% with respect the yield achieved in previous years, when no symptoms were observed. The symptoms were consistent with those previously reported for anthracnose on soybean caused by Colletotrichum chlorophyti, C. cliviae and C. gloeosporioides (Barbieri et al. 2017; Mahmodi et al. 2013; Yang et al. 2012). Diseased, 3-week old plants were collected. Small pieces, approximately 1 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (Song et al. 2020), containing chloramphenicol (50 µg/mL), under darkness at 28 °C for 3 days. Sequence of internal transcribed spacer (ITS), actin (ACT), ß-tubulin (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAP/span>DH) genes was performed as reported by Yang et al. (2015). Sequences were submitted to GenBank under accession numbers MT361074 (ITS) and MT415548-MT415550 (ACT, TUB2 and GAPDH). Blast search revealed that the amplified sequences had 100% (ITS; C. brevisporum TCHD, MH883805), 97.66% (ACT; C. brevisporum S38, KY986905), 99.06% (TUB2; C. brevisporum PF-2, KY705061) and 100% (GAPDH; C. brevisporum LJTJ27, KP823797) matches to multiple C. brevisporum strains, whereas all reported C. chlorophyti, C. cliviae and C. gloeosporioides strains showed no similarity to at least 2 of the studied genes. Molecular phylogenetic tree constructed using MEGA7 confirmed the identity of the pathogen. ACT and ITS sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest log likelihood (-1749.2186) is shown in Figure 1. The Colletotrichum strains previously found causing anthracnoseon soybean, and other relevant strains used in taxonomic analyses were included in the phylogenetic tree. Microscope observations showed the presence of 15-µm-long cylindrical conidia and septate mycelium, and agree with those reported for the morphology of C. brevisporum by Damm et al. (2019). To confirm pathogenicity, the mycelia from a 2 day-old culture on PDA was collected and suspended in sterile ddH2O (≈ 106 cells/mL) to prepare the inoculum. The pathogen was sprayed-inoculated on stem and leaves of healthy soybean plants. In control plants, sterile ddH2O was used. Inoculated plants were maintained in growth chamber at 28 °C and 50% relative humidity. Typical anthracnose symptoms were obsered 20 days after inoculation (Figure 2). C. brevisporum was reported to produce anthracnose on pumpkin, papaya, mulberry, coffee, passion fruit and pepper in China (Liu et al. 2017; Liu et al. 2019; Xue et al. 2019). Here, we report for the first time C. brevisporum causing anthracnose on soybean, an economically-relevant crop in China.

First Report of Botryosphaeria dothidea Causing Stem Canker on Soybean in China.

In September 2020, widespread stem canker on soybean (Glycine max) was detected in southeastern Jiangsu (Nantong municipality; 120.76° E, 32.23° N) in China. Mature plants, 14 weeks of cultivation, exhibited brown necrotic lesions and dried-up stem. The symptoms were observed in eleven soybean fields, 1.6 ha in total, and approximately 80% of the plants were symptomatic. The symptoms were consistent with those previously reported for stem canker on soybean caused by Diaporthe aspalathi, D. caulivora and D. sojae (Ghimire et al. 2019; Mena et al. 2020). Small pieces, approximately 0.4 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (PDA), containing chloramphenicol (50 µg/mL), under darkness at 28 ºC for 7 days. Amplification of internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and ß-tubulin (TUB2) genes was performed using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Jia et al. 2019). Sequences were submitted to GenBank under accession numbers MW130133 (ITS), MW147481 (EF1-α) and MW147482 (TUB2). Blast search revealed that the amplified sequences had 99.65% (ITS; B. dothidea JZB310202, MN945381), 100% (EF1-α; B. dothidea ZB-77, MH726166) and 99.75% (TUB2; B. dothidea ZB-1, MN642587) matches to multiple B. dothidea strains, whereas all reported Diaporthe strains showed no nucleotide identity to the amplified sequences. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. ITS, EF1-α and TUB2 sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-4291.3981) is shown in Figure 1. Diaporthe strains found causing stem canker on soybean, some Phytophthora sojae strains (which also cause dried-up stem on soybean) (Yang et al. 2019), and B. dothidea strains found in China in other hosts were included in the phylogenetic tree. To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem of 2-week old soybean plants. Mycelial plugs from a 7 day-old culture on PDA were placed on the wounds and covered with Parafilm. Sterilized PDA plugs were used as control. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity. Typical stem canker symptoms were observed 5 days after inoculation (Figure 2). Microscope observations showed the presence of septate mycelium, fusiform conidia and round conidiomata, and agreed with those previously reported for the morphology of B. dothidea strains (Phillips et al. 2013). During recent months, B. dothidea was reported to cause stem canker and leave wilt on a number of plant species in China (Huang et al. 2020; Ju et al. 2020; Wang et al. 2020a, 2020b, 2020c), confirming the expansion and host promiscuity of this pathogen. Stem canker on soybean has been thoroughly associated to Diaporthe strains; however, this is the first report of B. dothidea causing this disease. We believe that our results will help to better understand the pathogens affecting soybean production in China.

Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum.

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum.

Enhancement of nuclease P1 production by Penicillium citrinum YL104 immobilized on activated carbon filter sponge.

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

Germination time and in vitro gastrointestinal digestion impact on the isoflavone bioaccessibility and antioxidant capacities of soybean sprouts.

Soybeans' isoflavone content increases with germination; nevertheless, their bioaccessibility in the gastrointestinal system is limited. This study evaluated the influence of germination time (1, 3, 5, and 7 days) and in vitro gastrointestinal conditions on the isoflavone profile of soybean sprouts. The total isoflavones (4.07 mg/g) and the malonyl genistin (1.37 mg/g) had the highest contents on day 5 in the gastric phase. The highest isoflavone bioaccessibility was observed in daidzein, genistein, and glycitin. An increase in antioxidant capacity was found during germination (day 7 > day 5 > day 3); however, the same trend was not observed during in vitro digestion. In summary, the results indicate that soybean sprouts germinated for 5 days may be more beneficial for consumption since they have the highest and most readily absorbed levels of isoflavones. These data suggest that soybean sprouts may be a functional food that provides bioavailable antioxidants.

Dipicolinic acid reduces Epicoccum sorghinum symptoms on maize and inhibits tenuazonic acid biosynthesis.

BACKGROUND: Epicoccum sorghinum is a pathogenic fungus that causes leaf spot in a wide range of plants, including maize, and synthesizes the mycotoxin tenuazonic acid (TEA), which is carcinogenic. Despite the relevant economic and yield losses caused by E. sorghinum worldwide, methods for the control of this pathogen are lacking. RESULTS: In this work, the efficacy of Bacillus-produced dipicolinic acid (DPA) for control of E. sorghinum was evaluated using in vitro and in vivo assays, and compared with the efficacy of three commercial fungicides, including carbendazim, prochloraz, and thiram. DPA inhibited E. sorghinum mycelial growth, and conidia germination, and produced important alterations in E. sorghinum hyphae. Interestingly, 10 mM DPA reduced TEA biosynthesis by 86.6%. Although DPA rapidly degraded on maize leaves, 10 mM DPA showed higher preventive (67.4% lesion length inhibition) and inhibitory (89.5% lesion length inhibition) efficacies for the control of E. sorghinum on maize leaves compared to the commercial fungicides. CONCLUSIONS: Collectively, this study presents the first method for the control of E. sorghinum on maize and demonstrates that DPA application is a suitable approach to inhibit E. sorghinum symptoms in plants and TEA biosynthesis. © 2024 Society of Chemical Industry.

Enhancement of adenosine production by Bacillus subtilis CGMCC 4484 through metabolic flux analysis and simplified feeding strategies.

The objective of this research was to understand how the initial glucose concentration influences adenosine (AR) production and metabolic flux shift on the cultivation of Bacillus subtilis CGMCC 4484. Experiments confirmed that initial glucose concentration affects cell growth, AR production and metabolites, significantly. The flux distribution at the key nodes of glucose-6-phosphate (G6P), pyruvate (PYR) and acetyl coenzyme-A (AcCoA) could be affected by changing the glucose concentration. Based on kinetic analysis of specific rates, the low-glucose concentration was better for both cell growth and AR production during the first 12 h. However, the high-glucose concentration was more favorable for AR formation after 18 h. Furthermore, different simplified feeding strategies were designed to achieve higher AR accumulation. The final AR concentration of 15.60 g L(-1) was achieved when an optimized constant-feeding strategy was used, which was 21.02 % higher than batch fermentation. This was the first time to investigate the regulation of the glucose metabolism of AR-producing B. subtilis.

Occurrence of aflatoxins in water and decontamination strategies: A review.

Aflatoxins are highly carcinogenic metabolites produced by some Aspergillus species and are the most prevalent mycotoxins. Although aflatoxins are commonly synthesized during fungal colonization in preharvest maize, cereals, and nuts, they can be transported by rainfall to surface water and are a common toxin found in wastewater from some food industries. Here, the occurrence of aflatoxins in bodies of water is reviewed for the first time, along with the decontamination methods. Aflatoxins have been detected in surface, wastewater and drinking water, including tap and bottled water. The specific sources of water contamination remain unclear, which is an important gap that must be addressed in future research. Two main kinds of decontamination methods have been reported, including degradation and adsorption. The best degradation rates were observed using gamma and UV irradiations, oxidoreductases and ozone, while the best adsorption abilities were observed with minerals, polyvinyl alcohol, durian peel and activated carbon. Synthetic polymers could be used as membranes in pipes to remove aflatoxins in water flows. Although most decontamination methods were screened using AFB1, the other commonly found aflatoxins were not used in the screenings. Overall, the occurrence of aflatoxins in water could be a significant emerging public health concern largely ignored by local and international legislation. Numerous advances have been reported for the decontamination of aflatoxins in water; however, there is still a long way to go to put them into practice.

Antifungal and elicitor activities of p-hydroxybenzoic acid for the control of aflatoxigenic Aspergillus flavus in kiwifruit.

Aspergillus flavus not only reduces kiwifruit production but also synthesizes carcinogenic aflatoxins, resulting in a relevant threat to human health. p-Hydroxybenzoic acid (pHBA) is one of the most abundant phenolics in kiwifruit. In this study, pHBA was found to reduce A. flavus mycelial growth by blocking the fungal mitotic exit network (MEN) and cytokinesis and to inhibit the biosynthesis of aflatoxins B1 and B2. The application of pHBA promoted the accumulation of endogenous pHBA and induced oxidative stress in A. flavus-infected kiwifruit, resulting in an increase in H2O2 content and catalase (CAT) and superoxide dismutase (SOD) activities. Preventive and curative treatments with 5 mM pHBA reduced A. flavus advancement by 46.1% and 68.0%, respectively. Collectively, the antifungal and elicitor properties of pHBA were examined for the first time, revealing new insights into the role of pHBA in the defense response of kiwifruit against A. flavus infection.

p-Aminobenzoic acid inhibits the growth of soybean pathogen Xanthomonas axonopodis pv. glycines by altering outer membrane integrity.

BACKGROUND: p-Aminobenzoic acid (pABA) is an environmentally friendly bioactive metabolite synthesized by Lysobacter antibioticus. This compound showed an unusual antifungal mode of action based on cytokinesis inhibition. However, the potential antibacterial properties of pABA remain unexplored. RESULTS: In this study, pABA showed antibacterial activity against Gram-negative bacteria. This metabolite inhibited growth (EC50 = 4.02 mM), and reduced swimming motility, extracellular protease activity, and biofilm formation in the soybean pathogen Xanthomonas axonopodis pv. glycines (Xag). Although pABA was previously reported to inhibit fungal cell division, no apparent effect was observed on Xag cell division genes. Instead, pABA reduced the expression of various membrane integrity-related genes, such as cirA, czcA, czcB, emrE, and tolC. Consistently, scanning electron microscopy observations revealed that pABA caused major alternations in Xag morphology and blocked the formation of bacterial consortiums. In addition, pABA reduced the content and profile of outer membrane proteins and lipopolysaccharides in Xag, which may explain the observed effects. Preventive and curative applications of 10 mM pABA reduced Xag symptoms in soybean plants by 52.1% and 75.2%, respectively. CONCLUSIONS: The antibacterial properties of pABA were studied for the first time, revealing new insights into its potential application for the management of bacterial pathogens. Although pABA was previously reported to show an antifungal mode of action based on cytokinesis inhibition, this compound inhibited Xag growth by altering the outer membrane's integrity. © 2023 Society of Chemical Industry.

Dipicolinic acid enhances kiwifruit resistance to Botrytis cinerea by promoting phenolics accumulation.

BACKGROUND: Kiwifruit is highly susceptible to fungal pathogens, such as Botrytis cinerea, which reduce crop production and quality. In this study, dipicolinic acid (DPA), which is one of the main components of Bacillus spores, was evaluated as a new elicitor to enhance kiwifruit resistance to B. cinerea. RESULTS: DPA enhances antioxidant capacity and induces the accumulation of phenolics in B. cinerea-infected 'Xuxiang' kiwifruit. The contents of the main antifungal phenolics in kiwifruit, including caffeic acid, chlorogenic acid and isoferulic acid, increased after DPA treatment. DPA enhanced H2 O2 levels after 0 and 1 days, which promoted catalase (CAT) and superoxide dismutase (SOD) activities, reducing long-term H2 O2 levels. DPA promoted the up-regulation of several kiwifruit defense genes, including CERK1, MPK3, PR1-1, PR1-2, PR5-1 and PR5-2. Furthermore, DPA at 5 mM inhibited B. cinerea symptoms in kiwifruit (95.1% lesion length inhibition) more effectively than the commercial fungicides carbendazim, difenoconazole, prochloraz and thiram. CONCLUSIONS: The antioxidant properties of DPA and the main antifungal phenolics of kiwifruit were examined for the first time. This study uncovers new insights regarding the potential mechanisms used by Bacillus species to induce disease resistance. © 2023 Society of Chemical Industry.