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 VOCs, 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, while 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.

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.

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 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.

First Report of Bacterial Fruit Blotch caused by Kosakonia cowanii on Trichosanthis fructus in Jiangsu Province, China.

Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities, with water-soaked lesions (Fig 1). The affected T. fructus fruit (five weeks after blossom) were located in a field in Huai'an Municipality, Jiangsu Province (33.85°N, 119.00°E). The incidence was approximately 50%, causing great losses in fruit production. To isolate the causal agent, two symptomatic fruit from different plants were surface-disinfested with 75% (v/v) ethanol for 1 min, washed three times with sterile distilled water, and cultured on Nutrient agar (NA) plates at 28°C for 24 h. The obtained colonies were light yellow and transferred to fresh NA plates using the conventional repetitive streaking technique to obtain pure cultures. The purified bacterial cells were rod shaped, 1.64 to 2.47 µm long (n = 45), and 0.58 to 0.74 µm wide (n = 45) (Figure S2). Three isolates were used for further characterization. Biochemical tests indicated that the three isolates were Gram negative. DNA was extracted from the three bacterial isolates and used to amplify the16S rRNA (27F/1492R primers) and partial gyrB (UP1/Up2r primers) genes (Marchesi et al. 1998; Yamamoto and Harayama 1995). PCR products were purified using the DNA Clean-up Kit (CW2301, CWBIO), ligated into the PMD-19 vector (6013, Takara), and sequenced by Beijing Tsingke Biotech. The obtained 16S rRNA (GenBank accessions: OQ923996-OQ923998) and gyrB sequences (OR140942-OR140944) showed the best match, over 99%and 98% identity with 100% coverage to the K. cowanii type strain JCM 10956 (CP019445.1). To fulfill Koch's postulates, pathogenicity tests were conducted on healthy T. fructus fruit. T. fructus fruit showed no wounds or lesions, and were surface disinfected with 75% alcohol. The three isolates were grown in nutrient broth at 200 rpm in 28 oC for 24 h and re-suspended in sterilized ddH2O to OD600 = 0.6~1.0 (108~109cfu/mL). Five µL of bacterial suspension was inoculated into the healthy fruit surface with a sterile knife. For the control experiment, the same volume of sterilized ddH2O was used. In each treatment, four healthy T. fructus fruit were treated. All samples were incubated at 25°C and 75% humidity in a plant incubator (Bluepard, MGC-350BP-2). After 12 days, bacterial fruit blotch symptoms were observed in all the inoculated fruit. The pathogen was recovered from the infected fruit, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis. To further investigate the pathogenicity, four-week-old T. fructus plant leaves were inoculated with the above three isolated suspension (OD600=0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). Sterilized ddH2O was used as mock control. After 10 days, bacterial blight symptoms were observed in all inoculated leaves. To our knowledge, this is the first report of K. cowanii causing fruit blotch on T. fructus worldwide. This species has been previously associated with acute cholecystitis in humans (Berinson et al. 2020; Petrzik et al. 2021), but it was recently identified as the causal agent of bacterial wilt on patchouli, bacterial blight on soybean, and stalk rot in foxtail millet (Han et al. 2023; Krawczyk and Borodynko-Filas 2020; Zhang et al. 2022). China is the largest producer of T. fructus. This report reveals that K. cowanii has a greater host range than was known. This report will help to better understand the pathogens that affects T. fructus production in China.

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.

First Report of Bacterial Leaf Blight caused by Enterobacter asburiae on Sorghum in Jiangsu Province, China.

Sorghum (Sorghum bicolor [L.] Moench) is a major cereal crop in China, with a planting area of more than 674666 ha in 2021. In August 2022, bacterial leaf blight symptoms were observed on sorghum plants grown in a field in Huai'an (119.30437 ºE, 33.999644 ºN), in Jiangsu Province (Fig. 1). To determine the causal agent, four symptomatic leaves from different plants were surface sterilized with 75% (v/v) ethanol for 1 min and washed three times with ddH2O. The surface-sterilized plant tissues were cut into small pieces (4 × 4 mm in size) and cultured on Nutrient Agar (NA) plates at 28ºC for 24 h. To obtain pure cultures, these colonies were transferred to fresh NA plates by using the conventional streak plate method. The purified bacterial cells were rod-shaped, from 1.14 to 1.66 µm long, and from 0.61 to 0.86 µm wide (number of observations = 31) (Fig. 2). Three isolates were used for further characterization. The Gram stain test indicated that the three isolates were Gram negative. 16S rRNA (27F/1492R primers) and gyrB (UP1/Up2r) genes were amplified and sequenced (Marchesi et al. 1998; Yamamoto and Harayama 1995). The obtained 16S rRNA (0R143361-0R143363) and gyrB sequences (0R146993-0R146995) were submitted to GenBank. The 16S rRNA sequences of the three isolated strains showed over 98% identity (1447/1462, 1438/1462 and 1443/1460 bp) to the E. asburiae reference strains ENIPBJ CG1, CAV1043 and 1808 013 (CP014993.1, CP011591.1 and AP019632.1, respectively). Similarly, the gyrB sequences of the three strains showed 98% identity (1103/1129, 1105/1129 and 1108/1129 bp) to the same E. asburiae reference strains. Four-week-old sorghum plants were used in the pathogenicity tests. A phylogenetic tree was constructed with reference strains (Hoffmann et al., 2005). The healthy leaves were inoculated with bacterial suspensions of the three bacterial isolates (OD600 = 0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). For the control group, sterilized ddH2O was used. Each isolate was inoculated in three healthy plants. Inoculated plants were incubated at 28ºC and 75% humidity with alternating 12-h light and 12-h dark cycles with a photon flux density of 200 mmol/m2/s. After 10 days, bacterial leaf blight symptoms were observed in all the inoculated leaves. The inoculated leaves showed severe browning near the inoculation site (1-2 cm), and advanced yellowing from 2 to 7 cm from the inoculation site, while no symptoms were found in control group. The pathogen was recovered from the infected leaves, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis, fulfilling Koch's postulates (Fig 2). To our knowledge, this is the first report of E. asburiae causing bacterial leaf blight on sorghum worldwide. This species is a well-known pathogen of humans that can cause nosocomial infections (Markovska et al. 2019; Zhu et al. 2017). Recently, E. asburiae was identified as the causal agent of bacterial blight on rice and tuber rot on radish (Wang et al. 2023; Yu et al. 2021). The emergence E. asburiae as a plant pathogen may be produced by the numerous resistant strains reported during recent years. Pantoea ananatis has been reported as a common companion pathogen of E. asburiae (Xue et al. 2021). This report will help to better understand the host promiscuity of E. asburiae and reveals a new pathogen that affects sorghum production in China. This study also serves as a basis for future studies to develop management strategies and cultivation for the disease to prevent sorghum yield loss. As far as we know, no control method for the management of this new plant pathogen was reported to date, which highlights the potential hazard of this discovery. Reference Hoffmann, H., et al. 2005. Syst. Appl. Microbiol. 28:196. Kauffman, H. E., et al. 1973. Plant Dis. Rep. 57:537. Marchesi, J. R., et al. 1998. Appl. Environ. Microbiol. 64:795. Markovska, R., et al. 2019. Infect. Dis. 51:627. Wang, R., et al. 2023. Plant Dis. in press. https://doi.org/10.1094/PDIS-11-22-2650-PDN Xue, Y., et al. 2021. Plant Dis. 105:2078. Yamamoto, S., et al. 1995. Appl. Environ. Microbiol. 61:1104. Yu, L., et al. 2021. Plant Dis. 106:310. Zhu, B., et al. 2017. J. Glob. Antimicrob. Resist. 8:104.

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.

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 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.

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.

Molecular Cloning and Characterization of a Serotonin N-Acetyltransferase Gene, xoSNAT3, from Xanthomonas oryzae pv. oryzae.

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the top ten bacterial plant diseases worldwide. Serotonin N-acetyltransferase (SNAT) is one of the key rate-limiting enzymes in melatonin (MT) biosynthesis. However, its function in pathogenic bacteria remains unclear. In this study, a Xoo SNAT protein (xoSNAT3) that showed 27.39% homology with sheep SNAT was identified from a collection of 24 members of GCN5-related N-acetyltransferase (GNAT) superfamily in Xoo. This xoSNAT3 could be induced by MT. In tobacco-based transient expression system, xoSNAT3 was found localized on mitochondria. In vitro studies indicated that xoSNAT3 showed the optima enzymatic activity at 50 °C. The recombinant enzyme showed Km and Vmax values of 709.98 µM and 2.21 nmol/min/mg protein, respectively. Mutant △xoSNAT3 showed greater impaired MT biosynthesis than the wild-type strain. Additionally, △xoSNAT3 showed 14.06% less virulence and 26.07% less biofilm formation. Collectively, our results indicated that xoSNAT3 services as a SNAT involved in MT biosynthesis and pathogenicity in Xoo.

Identification of New Fusarium sulawense Strains Causing Soybean Pod Blight in China and Their Control Using Carbendazim, Dipicolinic Acid and Kojic Acid.

Soybean plants are highly susceptible to Fusarium species, which significantly reduce soybean production and quality. Several Fusarium species have been reported to synthesize mycotoxins, such as trichothecene, which have been related to major human diseases. In November 2021, soybean pods in Nantong municipality, China, showed black necrotic lesions during the harvest stage. The disease incidence reached 69%. The pathogen was identified as Fusarium sulawense via morphological analysis and sequencing of ITS, EF1-α and RPB2 genes. A PCR assay with primers targeting the trichothecene biosynthesis genes suggested that the three isolates could synthesize trichothecenes. The effectiveness of fungicide carbendazim and natural metabolites dipicolinic acid and kojic acid was screened for the management of F. sulawense on postharvest soybean pods. The highest efficacy was obtained when combining 3.8 mg/mL carbendazim and 0.84 mg/mL dipicolinic acid (curative efficacy: 49.1% lesion length inhibition; preventive efficacy: 82.7% lesion length inhibition), or 1.9 mg/mL carbendazim and 0.71 mg/mL kojic acid (preventive efficacy: 84.9% lesion length inhibition). Collectively, this report will lead to a better understanding of the safety hazards found in soybean products in China and reveals the application of dipicolinic and kojic acids to reduce the use of carbendazim.

Peel Diffusion and Antifungal Efficacy of Different Fungicides in Pear Fruit: Structure-Diffusion-Activity Relationships.

Fungal pathogens can invade not only the fruit peel but also the outer part of the fruit mesocarp, limiting the efficacy of fungicides. In this study, the relationships between fungicide structure, diffusion capacity and in vivo efficacy were evaluated for the first time. The diffusion capacity from pear peel to mesocarp of 11 antifungal compounds, including p-aminobenzoic acid, carbendazim, difenoconazole, dipicolinic acid, flusilazole, gentamicin, kojic acid, prochloraz, quinolinic acid, thiophanate methyl and thiram was screened. The obtained results indicated that size and especially polarity were negatively correlated with the diffusion capacity. Although some antifungal compounds, such as prochloraz and carbendazim, were completely degraded after a few days in peel and mesocarp, other compounds, such as p-aminobenzoic acid and kojic acid, showed high stability. When applying the antifungal compounds at the EC50 concentrations, it was observed that the compounds with high diffusion capacity showed higher in vivo antifungal activity against Alternaria alternata than compounds with low diffusion capacity. In contrast, there was no relationship between stability and in vivo efficacy. Collectively, the obtained results indicated that the diffusion capacity plays an important role in the efficacy of fungicides for the control of pear fruit diseases.

Exogenous genistein enhances soybean resistance to Xanthomonas axonopodis pv. glycines.

BACKGROUND: Xanthomonas axonopodis pv. glycines (Xag) is the causal agent of bacterial pustule disease and results in enormous losses in soybean production. Although isoflavones are known to be involved in soybean resistance against pathogen infection, the effects of exogenous isoflavones on soybean plants remain unexplored. RESULTS: Irrigation of soybean plants with isoflavone genistein inhibited plant growth for short periods, probably by inhibiting the tyrosine (brassinosteroids) kinase pathway, and increased disease resistance against Xag. The number of lesions was reduced by 59%-63% when applying 50 µg ml-1 genistein. The effects on disease resistance were observed for 15 days after treatment. Genistein also enhanced the disease resistance of soybean against the fungal pathogen Sclerotinia sclerotiorum. Exogenous genistein increased antioxidant capacity, decreased H2 O2 level and promoted the accumulation of phenolics in Xag-infected soybean leaves. Exogenous genistein reduced the amounts of endogenous daidzein, genistein and glycitein and increased the concentration of genistin, which was found to show strong antibacterial activity against the pathogen and to reduce the expression of virulence factor yapH, and flagella formation gene flgK. The expression of several soybean defense genes, such as chalcone isomerase, glutathione S-transferase and 1-aminocyclopropane-1-carboxylate oxidase 1, was upregulated after genistein treatment. CONCLUSIONS: The effects of exogenous genistein on soybean plants were examined for the first time, revealing new insights into the roles of isoflavones in soybean defense and demonstrating that irrigation with genistein can be a suitable method to induce disease resistance in soybean plants. © 2022 Society of Chemical Industry.

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.

Biocontrol Ability of the Bacillus amyloliquefaciens Group, B. amyloliquefaciens, B. velezensis, B. nakamurai, and B. siamensis, for the Management of Fungal Postharvest Diseases: A Review.

The Bacillus amyloliquefaciens group, composed of B. amyloliquefaciens, B. velezensis, B. nakamurai, and B. siamensis, has recently emerged as an interesting source of biocontrol agents for the management of pathogenic fungi. In this review, all the reports regarding the ability of these species to control postharvest fungal diseases have been covered for the first time. B. amyloliquefaciens species showed various antifungal mechanisms, including production of antifungal lipopeptides and volatile organic compounds, competition for nutrients, and induction of disease resistance. Most reports discussed their use for the control of fruit diseases. Several strains were studied in combination with additives, improving their inhibitory efficacies. In addition, a few strains have been commercialized. Overall, studies showed that B. amyloliquefaciens species are a suitable environmentally friendly alternative for the control of postharvest diseases. However, there are still crucial knowledge gaps to improve their efficacy and host range.

Antifungal Mechanism and Efficacy of Kojic Acid for the Control of Sclerotinia sclerotiorum in Soybean.

Sclerotinia stem rot, which is caused by the fungal pathogen Sclerotinia sclerotiorum, is a soybean disease that results in enormous economic losses worldwide. The control of S. sclerotiorum is a difficult task due to the pathogen's wide host range and its persistent structures, called sclerotia. In addition, there is lack of soybean cultivars with medium to high levels of resistance to S. sclerotiorum. In this work, kojic acid (KA), a natural bioactive compound commonly used in cosmetic industry, was evaluated for the management of Sclerotinia stem rot. Interestingly, KA showed strong antifungal activity against S. sclerotiorum by inhibiting chitin and melanin syntheses and, subsequently, sclerotia formation. The antifungal activity of KA was not obviously affected by pH, but was reduced in the presence of metal ions. Treatment with KA reduced the content of virulence factor oxalic acid in S. sclerotiorum secretions. Preventive applications of 50 mM KA (7.1 mg/ml) completely inhibited S. sclerotiorum symptoms in soybean; whereas, in curative applications, the combination of KA with prochloraz and carbendazim improved the efficacy of these commercial fungicides. Taken together, the antifungal activity of KA against S. sclerotiorum was studied for the first time, revealing new insights on the potential application of KA for the control of Sclerotinia stem rot in soybean.