SIRT5-Mediated Desuccinylation of RAB7A Protects Against Cadmium-Induced Alzheimer's Disease-Like Pathology by Restoring Autophagic Flux.

Cadmium (Cd) is a neurotoxic contaminant that induces cognitive decline similar to that observed in Alzheimer's disease (AD). Autophagic flux dysfunction is attributed to the pathogenesis of AD, and this study aimed to investigate the effect of autophagy on environmental Cd-induced AD progression and the underlying mechanism. Here, Cd exposure inhibited autophagosome-lysosome fusion and impaired lysosomal function, leading to defects in autophagic clearance and then to APP accumulation and nerve cell death. Proteomic analysis coupled with Ingenuity Pathway Analysis (IPA) identified SIRT5 as an essential molecular target in Cd-impaired autophagic flux. Mechanistically, Cd exposure hampered the expression of SIRT5, thus increasing the succinylation of RAB7A at lysine 31 and inhibiting RAB7A activity, which contributed to autophagic flux blockade. Importantly, SIRT5 overexpression led to the restoration of autophagic flux blockade, the alleviation of Aß deposition and memory deficits, and the desuccinylation of RAB7A in Cd-exposed FAD4T mice. Additionally, SIRT5 levels decrease mainly in neurons but not in other cell clusters in the brains of AD patients according to single-nucleus RNA sequencing data from the public dataset GSE188545. This study reveals that SIRT5-catalysed RAB7A desuccinylation is an essential adaptive mechanism for the amelioration of Cd-induced autophagic flux blockade and AD-like pathogenesis.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

First Report of Pectobacterium polaris Causing Soft Rot on Broccoli in China.

In April 2023, soft rot symptoms were observed in broccoli (Brassica oleracea L. var. italica) commercial fields in Songming County, Yunnan province, China (103°12'E, 25°31'N). The disease incidence in these fields (6 ha in size) was high, exceeding 50%, and it caused significant yield loss. The affected plants displayed characteristic symptoms, with the roots and stems of broccoli becoming soft, yellowish-brown, rotten, and emitting a foul odor. To identify the causal agent, soft rot symptomatic stems were surface sterilized by dipping them in 75% ethanol for 30 seconds, followed by three successive rinses with sterile distilled water. Tissue specimens were then plated onto nutrient agar (NA) plates and incubated at 28°C for 24 hours. (Wang et al. 2022). Three representative bacterial isolates HYC22041801-HYC22041803 from broccoli were selected for further analysis. The colonies on NA plates appeared as white, small, round, and translucent with smooth edges. Physiological and biochemical tests were performed, along with 96 phenotypic screenings using the BIOLOG GENIII microplate system (Biolog, Hayward, CA, USA). Three isolates were negative for D-arabitol, maltose, and sorbitol, but were positive for cellobiose, α-D-glucose, sucrose, glycerol and gentiobiose tests, which are consistent with the reported type strain P. polaris NIBIO1006T (Chen et al. 2021). Total genomic DNA was extracted from three bacterial isolates using the QIAamp DNA Mini Kit (QIAGEN, USA). The 16S rRNA region and nine housekeeping genes (gapA, icdA, mdh, mtlD, pel, pgi, pmrA, proA and rpoS) were amplified with universal primers 27F/1492R (Monciardini et al., 2006) and designed specific primers (Xie et al., 2018), respectively. All amplicons were sequenced and deposited in GenBank with accession numbers ON723841-ON723843 and ON723846-ON723872. The BLASTn analysis of the 16S rRNA amplicons confirmed that the isolates HYC22041801-HYC22041803 belonged to the genus Pectobacterium. Phylogenetic trees based on 16S rRNA gene sequences and multilocus sequence analysis of other nine housekeeping genes of the three isolates were constructed and the results revealed that three isolates clustered with P. polaris type strain NIBIO1006T, which was previously isolated from potato (Dees et al., 2017). To confirm the pathogenicity, nine broccoli seedlings were stab inoculated with a bacterial suspension (108 CFU·ml-1), while sterile distilled liquid LB medium was used as a negative control. The seedlings were kept at 80% relative humidity and 28°C in a growth chamber. Three trials were conducted per isolate (HYC22041801-HYC22041803). After 3 days, the inoculated petioles showed soft rot symptoms similar to those observed initially in the field, while control plants remained asymptomatic. All three isolates were re-isolated successfully from symptomatic tissues to complete Koch's postulates. P. polaris has been previously reported as the causative agent of blackleg in potato in several countries, including Norway, Poland, Russia, and China (Handique et al. 2022; Wang et al. 2022). Additionally, it was reported to cause soft rot in Chinese cabbage in China (Chen et al. 2021). However, this is the first report of P. polaris causing soft rot disease in broccoli in China. This discovery is of great importance for vegetable growers because this bacterium is well established on Cruciferous vegetables in the local area, and effective measures are needed to manage this disease.

Near-infrared imaging of head and neck squamous cell carcinoma using indocyanine green that targets the αvß6 peptide.

Significance: Head and neck squamous cell carcinoma (HNSCC) has a particularly poor prognosis. Improving the surgical resection boundary, reducing local recurrence, and ultimately ameliorating the overall survival rate are the treatment goals. Aim: To obtain a complete surgical resection (R0 resection), we investigated the use of a fluorescent imaging probe that targets the integrin subtype αvß6, which is upregulated in many kinds of epithelial cancer, using animal models. Approach: αvß6 expression was detected using polymerase chain reaction (PCR) and immunoprotein blotting of human tissues for malignancy. Protein expression localization was observed. αvß6 and epidermal growth factor receptor (EGFR) were quantified by PCR and immunoprotein blotting, and the biosafety of targeting the αvß6 probe material was examined using Cell Counting Kit-8 assays. Indocyanine green (ICG) was used as a control to determine the localization of the probe at the cellular level. In vivo animal experiments were conducted through tail vein injections to evaluate the probe's imaging effect and to confirm its targeting in tissue sections. Results: αvß6 expression was higher than EGFR expression in HNSCC, and the probe showed good targeting in in vivo and in vitro experiments with a good safety profile. Conclusions: The ICG-αvß6 peptide probe is an exceptional and sensitive imaging tool for HNSCC that can distinguish among tumor, normal, and inflammatory tissues.

First Report of Root Rot Caused by Fusarium incarnatum on Mongolian Snake gourd in China.

Mongolian snake gourd (Trichosanthes kirilowii Maxim) is a precious traditional Chinese herbal medicine and perennial liana plant in the family Cucurbitaceae, and the root, fruit, seed and peel all possess the medicinal value (Zhang et al. 2016). During 2021-2022, the root rot was observed in a 20-ha commercial farm and became a major disease limiting Mongolian snake gourd production in Zhenjiang City, Jiangsu Province, China (119°27'E, 32°12'N). Field investigations showed that disease incidence was estimated at approximately 70% and resulted in up to a 50% decrease in total production. Symptoms on snake gourd initially appeared as yellow mottling produced on the surface of the infected new leaves and systemic wilting on the upper leaves. With the development of the infection, the base of the stem began to brown and die, and has lots of filamentous hyphae attached to it. As the lesions coalesced, the whole plant gradually wilted and died. In order to explore the cause of the disease, six infected plants were randomly collected from the commercial farm. The roots of the plants were rinsed in sterile water to remove soil debris, and symptomatic roots were surface sterilized using 75% ethanol for 60s, rinsed three times in sterile water, then plated onto the potato dextrose agar (PDA), and incubated at 25°C for 3 days in the dark. White fungal colonies grew from the tissue pieces, then hyphal tips were transferred to PDA to obtain pure cultures. A total of six isolates with similar morphological characteristics were obtained from six of the infected plants. One representative isolate GL21091501 was chosen for further analysis. At 5 days after inoculation, the colonies on PDA began to grow as white, and with the incubated time was extended, the hyphae turned yellowish-brown with a yellowish-brown center on the reverse side. Observations under a light microscope showed conidia that were falculate, slender and slightly curved, and the cells at both ends were sharp. Macroconidia had four to five septa, measuring 22.4 ~ 33.5 µm. Microconidia without septa, elliptical, measuring 4.36 ~ 9.88 µm. On the tip of aerial hyphae can form conidiophore, and produce macroconidia (Wonglom et al. 2020; Lin et al 2018). The pathogen was typical Fusarium spp. by morphological characteristics. To identify the species level, the mycelia of the representative isolate GL21091501 was used for genomic DNA extraction (Tiangen, China). The internal transcribed spacer (ITS) region and partial translational elongation factor subunit 1-α (TEF-1α) of the cultures were amplified and sequenced using the primer pairs EF1/EF2 and ITS1/ITS4 respectively (White et al. 1990; O'Donnell et al. 1998). The obtained sequences were deposited in GenBank under the accesion numbers OP311409 and OP311410. BLAST searches of the deposited sequences showed 100% identity with the existing TEF sequences (MT563420.1) and ITS sequences (MN539094.1) of Fusarium incarnatum isolates in GenBank. In addition, BLASTn analysis of these in FUSARIUM-ID database showed 99.62% and 100% similarity with F. incarnatum-equiseti species complex (FIESC) NRRL13379 [ITS] and NRRL34004 [TEF-1α]), respectively. Phylogenetic analysis was conducted with the neighbor-joining (NJ) method using MEGA6.0 (Tamura et al. 2007). Combined phylogenetic analysis revealed that the isolate shared a common clade with the reference sequence of F. incarnatum in the F. incarnatum-equiseti species complex. Therefore, according to morphological and molecular characteristics confirming the identity of the isolated pathogen as F. incarnatum. In order to fulfill Koch's postulates, fresh isolate GL21091501 hyphae were cut into 3 × 3 mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL the Potato Dextrose (PD) liquid medium on a shaker at 170 rpm, 25°C for 5 days. Spores were filtered through four layers of gauze, adjusted to 1 × 106 spores/ml with sterilized water. Then Mongolian snake gourd seedlings at the two true leaves stage were transplanted in (15-cm-diameter) pots (1 plants/pot) filled with mixture of sterilized soil: vermiculite: pearlite (2:1:1, v/v). The pathogenicity test was conducted on seedlings plants by root irrigation method (50 ml/plant, 1×106 conidia/mL), control plants were irrigation with sterilized water (50 ml/plant). Each treatment was repeated three times. After 15 days, all inoculated plants showed the same symptoms observed on the original diseased plants in the field, whereas, the control plants remained symptomless. The same pathogen was successfully re-isolated from the inoculated plants, and identical to those of the originals based on morphological and sequence data. To our knowledge, this is the first report of F. incarnatum causing root rot on Mongolian snake gourd in China. F. incarnatum has been reported to cause root and stem rot in many plants worldwide, including muskmelon (Wonglom et al. 2020), Cucurbita pepo (Thomas et al. 2019) and Bambusa multiplex (Lin et al. 2018). This discovery is of great importance for Mongolian snake gourd planters because the fungus is accurately identified in a certain geographic area and effective field management strategies are necessary to control this disease.

Application of ultra-low-volume spray for the control of vegetable disease in greenhouse: Investigation of formulation performance and potential dermal exposure.

BACKGROUND: The use of pesticides in greenhouse vegetable cultivation is necessary and significant. However, traditional pesticide application methods such as the use of backpack sprayers with water-diluted pesticides have certain drawbacks, e.g., uneven distribution, high labor intensity, and safety risks. RESULTS: In this work, fluazinam ultra-low-volume liquids (Flu-ULVs) were prepared using oily solvents as carriers. The effects of different oils on the physical properties of the preparations were investigated. The Flu-ULV can be sprayed directly using a hand-held ultra-low-volume (ULV) sprayer without dilution with water. Compared with commercial water-based suspension concentrates of fluazinam, the Flu-ULV samples showed better wetting of tomato leaves, better atomization, and more uniform droplet distribution. At a dosage of 300 mL/ha, the coverage rate of tomato leaves ranged from 32.47% to 79.3%, with a droplet deposition density of 556 to 2017 droplets/cm2. Application of Flu-ULVs provided 70.86% control efficacy against gray mold in tomatoes, which was higher than those achieved with reference products. Dermal exposure to Flu-ULVs was also evaluated in greenhouse experiments. The coverage rates for all parts of the operator's body ranged from 0.02% to 0.07%, with deposition volumes of 2.23 to 12.26 µg/cm2. CONCLUSION: Ground ULV spraying of fluazinam was proved to be an effective and safe management option for the control of tomato gray mold in greenhouses. This study laid a foundation for the use of ultra-low volume spray to control vegetable diseases in greenhouse, especially those induced by high humidity environment. © 2024 Society of Chemical Industry.

Deciphering Differences in Microbial Community Diversity between Clubroot-Diseased and Healthy Soils.

Clubroot (Plasmodiophora brassicae) is an important soilborne disease that causes severe damage to cruciferous crops in China. This study aims to compare the differences in chemical properties and microbiomes between healthy and clubroot-diseased soils. To reveal the difference, we measured soil chemical properties and microbial communities by sequencing 18S and 16S rRNA amplicons. The available potassium in the diseased soils was higher than in the healthy soils. The fungal diversity in the healthy soils was significantly higher than in the diseased soils. Ascomycota and Proteobacteria were the most dominant fungal phylum and bacteria phylum in all soil samples, respectively. Plant-beneficial microorganisms, such as Chaetomium and Sphingomonas, were more abundant in the healthy soils than in the diseased soils. Co-occurrence network analysis found that the healthy soil networks were more complex and stable than the diseased soils. The link number, network density, and clustering coefficient of the healthy soil networks were higher than those of the diseased soil networks. Our results indicate that the microbial community diversity and network structure of the clubroot-diseased soils were different from those of the healthy soils. This study is of great significance in exploring the biological control strategies of clubroot disease.

Development of chitosan/carrageenan macrobeads for encapsulation of Paenibacillus polymyxa and its biocontrol efficiency against clubroot disease in Brassica crops.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most important diseases of brassicas. The antagonistic bacterium Paenibacillus polymyxa ZF129 can suppress clubroot while its effectiveness is often unstable. To control clubroot more effectively, the macrobeads for controlled release of ZF129 were prepared using microencapsulation technology. Macrobeads with various ratios of chitosan (2 % w/w): carrageenan (0.3 % w/v) were prepared by an ionotropic gelation method and the bacteria ZF129 was loaded into macrobeads. The 1:1 chitosan: carrageenan showed the maximum swelling ratio (634 %), and the maximum survival rate (61.52 ± 1.12 %) after freeze-drying. Fourier transform infrared revealed the electrostatic interactions between chitosan and carrageenan. The macrobeads can efficiently release ZF129 strains into phosphate buffer solution and reach equilibrium in 48 h. The maximum number of bacteria cells to be released in the soil was observed after 25-30 days. The control efficacy of ZF129 macrobeads (chitosan: carrageenan, 1:1) and ZF129 culture against clubroot disease was 76.33 ± 3.65 % and 59.76 ± 4.43 % in greenhouse experiments, respectively and the control efficacy was calculated as 60.74 ± 5.00 % for ZF129 macrobeads and 40.94 ± 4.05 % for ZF129 culture under field experiments, respectively. The ZF129 macrobeads had significant growth-promoting effects on pak choi and Chinese cabbage. The encapsulation method described in this study is a prudent approach toward efficient biopesticides utilization with reduced environmental implications.

The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 µg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 µg/mL), and highly resistant isolates (HR, EC50 > 56.98 µg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

Monitoring Corynespora cassiicola Resistance to Boscalid, Trifloxystrobin, and Carbendazim in China.

Cucumber leaf spot (CLS), caused by Corynespora cassiicola, is a serious disease of greenhouse cucumbers. With frequent use of existing fungicides, C. cassiicola has developed resistance to some of them, with serious implications for the control of CLS in the field. With a lack of new fungicides, it is necessary to use existing fungicides for effective control. Therefore, this study monitored the resistance of C. cassiicola to three commonly used and effective fungicides, boscalid, trifloxystrobin, and carbendazim, from 2017 to 2021. The frequency of resistance to boscalid showed an increasing trend, and the highest frequency was 85.85% in 2020. The frequency of resistance to trifloxystrobin was greater than 85%, and resistance to carbendazim was maintained at 100%. Among these fungicides, strains with multiple resistance to boscalid, trifloxystrobin, and carbendazim were found, accounting for 32.00, 25.25, 33.33, 43.06, and 37.24%, respectively. Of the strains that were resistant to boscalid, 87% had CcSdh mutations, including seven genotypes: B-H278L/Y, B-I280V, C-N75S, C-S73P, D-D95E, and D-G109V. Also, six mutation patterns of the Ccß-tubulin gene were detected: E198A, F167Y, E198A&M163I, E198A&F167Y, M163I&F167Y, and E198A&F200C. Detection of mutations of the CcCytb gene in resistant strains showed that 98.8% were found to have only the G143A mutation. A total of 27 mutation combinations were found and divided into 14 groups for analysis. The resistance levels differed according to genotype. The development of genotypes showed a complex trend, increasing from 4 in 2017 to 13 in 2021 and varying by region. Multiple fungicide resistance is gradually increasing. Therefore, it is necessary to understand the types of mutations and the trend of resistance to guide the use of fungicides to achieve disease control.

Transcriptome Analysis Reveals the Involvement of Mitophagy and Peroxisome in the Resistance to QoIs in Corynespora cassiicola.

Quinone outside inhibitor fungicides (QoIs) are crucial fungicides for controlling plant diseases, but resistance, mainly caused by G143A, has been widely reported with the high and widespread use of QoIs. However, two phenotypes of Corynespora casiicola (RI and RII) with the same G143A showed significantly different resistance to QoIs in our previous study, which did not match the reported mechanisms. Therefore, transcriptome analysis of RI and RII strains after trifloxystrobin treatment was used to explore the new resistance mechanism in this study. The results show that 332 differentially expressed genes (DEGs) were significantly up-regulated and 448 DEGs were significantly down-regulated. The results of GO and KEGG enrichment showed that DEGs were most enriched in ribosomes, while also having enrichment in peroxide, endocytosis, the lysosome, autophagy, and mitophagy. In particular, mitophagy and peroxisome have been reported in medicine as the main mechanisms of reactive oxygen species (ROS) scavenging, while the lysosome and endocytosis are an important organelle and physiological process, respectively, that assist mitophagy. The oxidative stress experiments showed that the oxidative stress resistance of the RII strains was significantly higher than that of the RI strains: specifically, it was more than 1.8-fold higher at a concentration of 0.12% H2O2. This indicates that there is indeed a significant difference in the scavenging capacity of ROS between the two phenotypic strains. Therefore, we suggest that QoIs' action caused a high production of ROS, and that scavenging mechanisms such as mitophagy and peroxisomes functioned in RII strains to prevent oxidative stress, whereas RI strains were less capable of resisting oxidative stress, resulting in different resistance to QoIs. In this study, it was first revealed that mitophagy and peroxisome mechanisms available for ROS scavenging are involved in the resistance of pathogens to fungicides.

Seed Pelleting with Gum Arabic-Encapsulated Biocontrol Bacteria for Effective Control of Clubroot Disease in Pak Choi.

Clubroot is one of the most serious soil-borne diseases on crucifer crops worldwide. Seed treatment with biocontrol agents is an effective and eco-friendly way to control clubroot disease. However, there is a big challenge to inoculating the seed with bacterial cells through seed pelleting due to the harsh environment on the seed surface or in the rhizosphere. In this study, a method for microbial seed pelleting was developed to protect pak choi seedlings against clubroot disease. Typically, a biocontrol bacterium, Paenibacillus polymyxa ZF129, was encapsulated by the spray-drying method with gum arabic as wall material, and then pak choi seeds were pelleted with the microencapsulated Paenibacillus polymyxa ZF129 (ZF129m). The morphology, storage stability, and release behavior of ZF129 microcapsules were evaluated. Compared with the naked Paenibacillus polymyxa ZF129 cells, encapsulated ZF129 cells showed higher viability during ambient storage on pak choi seeds. Moreover, ZF129m-pelleted seeds showed higher control efficacy (71.23%) against clubroot disease than that of nonencapsulated ZF129-pelleted seeds (61.64%) in pak choi. Seed pelleting with microencapsulated biocontrol Paenibacillus polymyxa ZF129 proved to be an effective and eco-friendly strategy for the control of clubroot disease in pak choi.

Rapid Detection and Quantification of Viable Cells of Pectobacterium brasiliense Using Propidium Monoazide Combined with Real-Time PCR.

Pectobacterium brasiliense (Pbr) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for Pbr by screening specific and sensitive primers based on the glu gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y = -3.391x + 36.28; R2 = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 102 CFU·mL-1. The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 102 to 104 CFU·g-1 viable Pbr bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by Pbr is crucial.

Integration of transcriptomics and metabolomics reveals a novel gene signature guided by FN1 associated with immune response in oral squamous cell carcinoma tumorigenesis.

PURPOSE: Oral squamous cell carcinomas (OSCCs) are primary head and neck malignant tumours with a high incidence and mortality. However, the molecular mechanisms involved in OSCC tumorigenesis are not fully understood. METHODS: OSCC and paired para-carcinoma samples were collected and used to perform multi-omics study. Transcriptomic analysis was used to reveal significant alterations in inflammatory and immune processes in OSCC. Ingenuity Pathway Analysis (IPA) combined with the LASSO Cox algorithm was used to identify and optimize a crucial gene signature. Metabolomics analysis was performed to identify the important metabolites which linked to the crucial gene signature. The public data TCGA-HNSCC cohort was used to perform the multiple bioinformatic analysis. RESULTS: These findings identified a FN1-mediated crucial network that was composed of immune-relevant genes (FN1, ACP5, CCL5, COL1A1, THBS1, BCAT1, PLAU, IGF2BP3, TNF, CSF2, CXCL1 and CXCL5) associated with immune infiltration and influences the tumour microenvironment, which may contribute to OSCC tumorigenesis and progression. Moreover, we integrated the relevant genes with altered metabolites identified by metabolic profiling and identified 7 crucial metabolites (Glu-Glu-Lys, Ser-Ala, Ser-Ala, N-(octadecanoyl) sphing-4-enine-1-phosphocholine, N-methylnicotinamide, pyrrhoxanthinol and xanthine) as potential downstream targets of the FN1-associated gene signature in OSCC. Importantly, FN1 expression is positively correlated with immune infiltration levels in HNSCC, which was confirmed at the single-cell level. CONCLUSIONS: Overall, these results revealed the differential genetic and metabolic patterns associated with OSCC tumorigenesis and identified an essential molecular network that plays an oncogenic role in OSCC by affecting amino acid and purine metabolism. These genes and metabolites might, therefore, serve as predictive biomarkers of survival outcomes and potential targets for therapeutic intervention in OSCC.

Calcium carbonate-modified plant sporopollen capsule as an eco-friendly microvehicle for controlled release of pesticide.

BACKGROUND: In this work, natural club moss (Lycopodium clavatum, LC) spores with a porous surface morphology and highly uniform size distribution were engineered into controlled-release microvehicles for pesticide delivery. As a proof of concept, a widely used fungicide, fluazinam (FLU), was successfully loaded into LC spores and then modified with different amounts of CaCO3 (CaC) to extend the efficacy duration of FLU. Significantly, as the control target of FLU, clubroot disease is a worldwide destructive disease of cruciferous crops, and its development is favored by acidic soils and can be suppressed at high Ca concentrations. RESULTS: Fabricated FLU@LC-CaC microcapsules, FLU loading and CaCO3 deposition were systematically characterized by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The as-prepared FLU@LC-CaC microcapsules showed sustained-release behaviors and were potentially able to supplement the Ca concentration in acidic environments. This approach synergistically enhanced in vivo bioactivity for the on-demand control of clubroot disease. An in vivo bioassay revealed that the control efficacy of FLU@LC-CaC against clubroot disease in pak choi (Brassica chinensis) (66.4%) was 1.7-fold higher than that of a commercial FLU suspension concentrate (38.2%) over the course of the cultivation period (35 days). CONCLUSIONS: This work provides new ideas not only for developing eco-friendly and scalable microvehicles for pesticide delivery based on natural sporopollen, but also for unconventional research perspectives in on-demand pest management based on their occurrence characteristics. © 2022 Society of Chemical Industry.

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of succinate dehydrogenase inhibitors (SDHIs), resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and eight resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern III (BosVHR, FluoSS, PenLR, CarS); pattern IV (BosLR, FluoLR, PenS, CarR); pattern V (BosMR, FluoLR, PenS, CarS); pattern VI (BosMR, FluoLR, PenLR, CarS); pattern VII (BosVHR, FluoHR, PenHR, CarS); and pattern VIII (BosLR, FluoLR, PenLR, CarS). We successfully established nine allele-specific PCR (AS-PCR) assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µl, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µl. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR, and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR (MAS-PCR) assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

The occurrence of clubroot in cruciferous crops correlates with the chemical and microbial characteristics of soils.

Clubroot disease, caused by Plasmodiophora brassicae, is a serious soil-borne disease in Brassica crops worldwide. It seriously occurs in conducive soils of southern China, while never happens in some areas of northern China with suppressive soils. To understanding the differences, we measured the soil suppressiveness, chemical properties, and microbial communities in suppressive and conducive soils by bioassay and sequencing of 16S and 18S rRNA amplicons. The biological basis of clubroot suppressiveness was supported by the ability to remove it by pasteurization. The pH value and calcium content in the suppressive soils were higher than those in the conducive soils. Suppressive soils were associated with higher fungal diversity and bacterial abundance. The fungal phyla Chytridiomycota, Olpidiomycota, and Mucoromycota and the bacterial phyla Acidobacteriota and Gemmatimonadota were enriched in suppressive soils. More abundant beneficial microbes, including Chaetomium and Lysobacter, were found in the suppressive soils than in the conducive soils. Molecular ecological network analysis revealed that the fungal network of suppressive soils was more complex than that of conducive soils. Our results indicate that plant health is closely related to soil physicochemical and biological properties. This study is of great significance for developing strategies for clubtroot disease prevention and control.

Temperature and Humidity Regulate Sporulation of Corynespora cassiicola That Is Associated with Pathogenicity in Cucumber (Cucumis sativus L.).

Cucumber target leaf spot, caused by Corynespora cassiicola, is an emerging disease with a high incidence that causes severe damage to cucumbers on a global scale. Therefore, efforts need to be undertaken to limit the spread and infection of this pathogen, preferably by using environmentally friendly methods. In this study, the effects of temperature and moisture on the sporulation of C. cassiicola were investigated in vitro and in vivo. The novelty of our study refers to the observation of spore production and size as well as the revelation of a correlation between spore size and virulence. On potato dextrose agar (PDA) and cucumber-leaf extract agar (CEA), temperature played a critical role in spore production, which was strongly influenced by both temperature and moisture on detached leaves and cucumber seedlings. Maximum spore production was found at 30 °C on PDA and 25 °C on CEA, cucumber detached leaves and living plants. Lower spore productions were observed with a stepwise change of 5 °C. In addition, the largest spore production was found at 100% relative humidity (RH) in comparison to the other tested moisture. Moreover, moisture was found to be the most important factor affecting spore size, accounting for 83.09-84.86% of the total variance in length and 44.72-73.10% of the total variance in width. The longest-narrowest spores were formed at 100% RH, and the shortest-widest spores were formed at 75% RH. Furthermore, the result showed that larger spores of C. cassiicola were more virulent and small spores were avirulent. Our findings will contribute to the development of new strategies for the effective alleviation and control of cucumber target leaf spot.

Rapid Quantification of Infectious Cucumber green mottle mosaic virus in Watermelon Tissues by PMA Coupled with RT-qPCR.

Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 102 copies·µL-1. PMA at 120 µmol·L-1 was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 102~105 copies·g-1. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources.