Coating seeds with biocontrol bacteria-loaded sodium alginate/pectin hydrogel enhances the survival of bacteria and control efficacy against soil-borne vegetable diseases.

Microbial seed coatings serve as effective, labor-saving, and ecofriendly means of controlling soil-borne plant diseases. However, the survival of microbial agents on seed surfaces and in the rhizosphere remains a crucial challenge. In this work, we embedded a biocontrol bacteria (Bacillus subtilis ZF71) in sodium alginate (SA)/pectin (PC) hydrogel as a seed coating agent to control Fusarium root rot in cucumber. The formula of SA/PC hydrogel was optimized with the highest coating uniformity of 90 % in cucumber seeds. SA/PC hydrogel was characterized using rheological, gel content, and water content tests, thermal gravimetric analysis, and Fourier transform infrared spectroscopy. Bacillus subtilis ZF71 within the SA/PC hydrogel network formed a biofilm-like structure with a high viable cell content (8.30 log CFU/seed). After 37 days of storage, there was still a high number of Bacillus subtilis ZF71 cells (7.23 log CFU/seed) surviving on the surface of cucumber seeds. Pot experiments revealed a higher control efficiency against Fusarium root rot in ZF71-SA/PC cucumber seeds (53.26 %) compared with roots irrigated with a ZF71 suspension. Overall, this study introduced a promising microbial seed coating strategy based on biofilm formation that improved performance against soil-borne plant diseases.

First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease on Broccoli in China.

Broccoli (Brassica oleracea L. var. italica) is one of the important cruciferous vegetables in China, known for its considerable economic and nutritional value (Li et al. 2021). In September 2023, leaf spot disease was observed on broccoli seedlings in the commercial fields in Weifang City, Shandong Province, China (119°15'E, 36°70'N). Further investigation revealed that the disease incidence was approximately 60% in 4.5 square hectometers (hm2) broccoli field, resulting in substantial economic losses. This disease primarily affects the leaves, manifesting distinct symptoms such as circular, dark necrotic spots that gradually lighten and are encircled by a chlorotic halo. Some lesions further develop a black or purplish border, exhibit concentric zonation, and eventually fall off, leaving behind holes, as shown in Figure S1B and C. In severe cases, decay originates from the central perforation and spreads outwards, as shown in Figure S1A. To identify the causal agent of this disease, infected leaf tissues were collected and surface disinfected by immersing in 75% ethanol for 30 seconds, followed by three rinses with sterile water. The samples were grinded in sterile deionized water, and the extract was plated on NA. After incubation at 28°C for 48 h, individual colonies were transferred to fresh NA plates. A total of 12 strains with the similar morphological characteristics were isolated from diseased samples collected from the three plots. After 48 hours of growth on NA medium at 28℃, each colony attained a diameter ranging from 3 to 5 mm. These colonies appeared yellow, slightly elevated, nearly circular in shape, with a smooth and moist edge. Three representative strains were selected for further investigation. All strains were Gram-negative, aerobic, and rod-shaped. The analysis of BIOLOG GENIII microplate system revealed the capability of three isolates using cellobiose, trehalose, glucose, mannose, galactose, and sucrose. Furthermore, the isolates were unable to hydrolyze arginine and utilize rhamnose and inositol. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Xanthomonas (OR772321-OR772323), followed by PCR amplification for 4 housekeeping genes atpD, dnaK, gyrB, and rpoD (Young et al. 2008; Saux et al. 2015). The obtained sequences were submitted to the GenBank under accessions OR789628-OR789630, OR785471-OR785473, OR789631-OR789633, and OR785468-OR785470. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). The phylogenetic analysis revealed that all the three isolates were clustered with Xanthomonas campestris pv. raphani strains 5055 and 576, respectively (Figure S2). (Dubrow et al. 2022). These results were consistent with those of the reported X. campestris pv. raphanin (Cruz et al. 2015). To verify the pathogenicity of these strains, we used a spray inoculation method. In detail, bacterial suspensions (30 mL per treatment) containing approximately 108 CFU/ml were sprayed onto healthy, four-week-old broccoli plants and incubated in a phytotron at 28°C and above 90% RH. Negative controls were performed using sterile distilled water. Each isolate underwent three trials and each treatment included 12 broccoli seedlings. Leaf spot symptoms were observed on 5 days post inoculation, as shown in Figure S1E, F and G. Negative control plants showed no symptoms (Figure S1D). We further re-isolated the bacterium from the symptomatic plants and verifying the bacteria as X. campestris pv. raphanin with the aforementioned sequence analysis, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of X. campestris pv. raphani causing leaf spot disease on broccoli in China. This study enhances our understanding of the pathogenic bacterium on broccoli and lays the groundwork for developing targeted management strategies.

Seed coating with biocontrol bacteria encapsulated in sporopollenin exine capsules for the control of soil-borne plant diseases.

Coating seeds with biocontrol agents represents an effective approach for managing soil-borne plant diseases. However, improving the viability of biocontrol microorganisms on the seed surface or in the rhizosphere remains a big challenge due to biotic and abiotic stresses. In this work, we developed a microbial seed coating strategy that uses sporopollenin exine capsules (SECs) as carriers for the encapsulation of the biofilm-like biocontrol bacteria. SECs was extracted from camellia bee pollen, and then characterized by Fourier Transform infrared spectroscopy (FTIR), elemental analysis and thermal gravity analysis (TG). The Paenibacillus polymyxa ZF129, a biocontrol bacterium, was introduced into SECs using the vacuum-incubation method and characterized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Notably, the ZF129 cell formed a biofilm-like structure inside the SECs, which enhanced their tolerance to acidic stress. As a proof of concept, we applied ZF129-loaded SECs to coat pak choi seeds using a straightforward plate-shaking technique. The coated seeds demonstrated a high control efficacy of up to 60.46 % against clubroot disease. Overall, this study sheds light on the application of SECs as promising carrier for the encapsulation of biofilm-like biocontrol bacteria, further augmenting the biocontrol functionality of microbial seed coating.

First Report of Gibellulopsis nigrescens Causing Yellow Wilt on Chinese Cabbage in China.

Chinese cabbage (Brassica rapa L. ssp. pekinensis), in the family Brassicaceae, is a widely planted crop in China valued for its nutritional benefits. In May 2023, wilt symptoms on Chinese cabbage (cv. 'Dongtian118') were observed in several commercial fields located in Sheqi County, (32.47ºN, 112.46ºE), Nanyang, Henan Province, China. A disease survey noted that disease incidence on plants was approximately 20% to 50% within observed fields. Symptoms included yellowing and wilting leaves, and vascular discoloration of the stem bases. To isolate the pathogen, ten symptomatic leaves collected from different diseased cabbage in two field were cut into small pieces (5 × 5 mm), surface disinfected with 75% ethanol for 30 s, then washed three times in sterile water. After drying, tissues were transferred onto potato dextrose agar (PDA). Plates were incubated at 28℃ for 7 days in the dark. Twelve morphologically similar fungal isolates were obtained by single-spore subculture. The mycelia on PDA were originally white, later becoming dark gray due to the formation of masses of melanized chlamydospores after 15 days of culture. Conidiophores were hyaline and most had secondary branches. In addition, verticillate branches had three to four phialides in each whorl. The conidia were hyaline, elliptical or nearly circular, measuring from 3.2 to 9.5 × 2.6 to 3.8 µm (n=40). These morphological characteristics were similar to those described for Gibellulopsis nigrescens (Zare et al. 2007). The isolates were further identified based on PCR amplification. The ITS, GAPDH, and TEF1 genes were amplified using primers ITS1/ITS4, VGPDf2/VGPDr (Inderbitzin et al. 2011) and EF-2/EF1-728F (O'Donnell et al. 1998). BLAST analysis revealed 12 isolates were highly similar to G. nigrescens, with 99.82% similarity for ITS (OR818474, KJ534578), 93.17% similarity for GAPDH (JN188192.1, JN188166.1) and 91.07% similarity for TEF1 (EF543798.1, EF543804.1). Sequences of the representative isolate BC230515 were deposited into NCBI GenBank with accession nos. OR889646 for ITS and PP135039 for GAPDH. Pathogenicity of all 12 isolates was tested on potted Chinese cabbage plants (cv. 'Dongtian118'). Twenty-four healthy Chinese cabbage plants were inoculated by applying a 10 mL conidial suspension (1×107 conidial/mL) at the artificially wounded root region of each plant. Twenty-four control plants wounded similarly were treated with sterile distilled water. All plants were kept in a growth chamber at 22~25°C (day)/18~20°C (night) , 85% relative humidity and a photoperiod of 12 h per day. After 15 days, inoculated plants exhibited wilting symptoms similar to those observed in the field, whereas control plants remained healthy. The pathogenicity test was repeated three times. The associated fungus on the artificially inoculated plants was reisolated from the symptomatic leaves, and its identity was confirmed by PCR with the primers described above. Reisolated G. nigrescens had identical morphological and molecular characteristics to the original isolates, confirming Koch's postulates. To our knowledge, this is the first report of G. nigrescens causing yellowing and wilt of Chinese cabbage in China. G. nigrescens is a destructive pathogen with multiple hosts such as beet (Zhou et al. 2017), alfalfa (Hu et al. 2011), prevention and control measures should be taken in advance.

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.

Identification and Genetic Diversity Analysis of the Pathogen of Anthracnose of Pepper in Guizhou.

Anthracnose of pepper is a significant disease caused by Colletotrichum spp. In 2017 and 2021, 296 isolates were obtained from 69 disease samples. Through morphological analysis, pathogenicity detection, and polygenic phylogenetic analysis, the above strains were attributed to 10 species: C. scovillei, C. fructicola, C. karstii, C. truncatum, C. gloeosporioides, C. kahawae, C. boninense, C. nymphaeae, C. plurivorum, and C. nigrum. C. scovillei had the most strains (150), accounting for 51.02% of the total isolates; C. fructicola came in second (72 isolates), accounting for 24.49%. Regarding regional distribution, Zunyi City has the highest concentration of strains-92 strains total, or 34.18%-across seven species. Notably, this investigation showed that C. nymphaeae infected pepper fruit for the first time in China. Genetic diversity analysis showed that C. fructicola could be divided into seven haplotypes, and the population in each region had apparent genetic differentiation. However, the genetic distance between each population was not significantly related to geographical distance. Neutral detection and nucleotide mismatch analysis showed that C. fructicola might have undergone population expansion.

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.

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.

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.

Simplification of the pretreatment method for phosphate oxygen isotope measurement in phosphogypsum leachate.

The stacking of phosphogypsum has caused considerable phosphorus pollution in water bodies near phosphogypsum yards through surface runoff and underground infiltration. The phosphate oxygen isotope (δ18Op) tracing method has served as a valuable tool for tracing phosphorus pollution in water. However, the existing δ18Op enrichment and purification methods are complex, costly, and inefficient for phosphate recovery, particularly for phosphogypsum leachate with complex compositions. Herein, a simplified and optimized pretreatment method for δ18Op measurement in phosphogypsum leachate was developed. Zirconium/polyvinyl alcohol (Zr/PVA) gel beads showed good selectivity for phosphate enrichment from water at different initial phosphate concentrations with appropriate Zr/PVA dosage. The optimal enrichment pH value was <7, and the concentrated phosphate on the Zr/PVA gel beads could be effectively eluted in an alkaline environment. Compared with the traditional Fe or Mg coprecipitation enrichment methods, impurities in the solution showed no obvious adverse effects on the phosphate enrichment process. Further, the phosphate solution eluted from the Zr/PVA gel beads was purified by a simple adjustment of the pH instead of cation exchange in the traditional purification process. Magnesium ions in the solution could be completely removed when the pH ranged from 3.17 to 6.15, and the phosphate recovery rate could reach 98.66% when the eluent pH was 5.02. Fourier-transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy revealed that similar to traditional pretreatment method, the proposed method can obtain high-purity Ag3PO4 solids for δ18OP measurement and no isotope fractionation of δ18OP was observed. Therefore, this study provides a promising and reliable pretreatment method for δ18OP measurement, especially in complex phosphogypsum leachate.

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.

Effect of temperature and humidity on dynamics and transmission of Pseudomonas amygdali pv. lachrymans aerosols.

Cucumber angular leaf spot (ALS) disease, caused by Pseudomonas amygdali pv. lachrymans (Pal), is an emerging disease with a high incidence that causes severe damage to cucumber worldwide. Bacterial aerosols play a crucial role in the epidemiology of greenhouse ALS disease. However, little is known about the influence of temperature and relative humidity (RH) on the dynamics of Pal in aerosols. A study was conducted to investigate the relationships between the concentration of Pal aerosols and their dependence on temperature and RH in aerosol chambers and greenhouses. The results demonstrated that temperature and RH are both significant factors influencing the release amount, survival time and infectivity of Pal in aerosols, while RH has a greater influence on particle size than temperature across the range of conditions tested. The release amount and survival time of Pal in aerosols under high RH (95%) and low temperature (≤ 25°C) conditions were significantly higher than those under low RH (35%) and high temperature (35°C) conditions. The highest release amount of Pal aerosol (96 CFU/m3) and highest survival rate (98.41%) were found at 18°C and 95% RH, while the highest disease index (DI = 60.9) caused by Pal aerosol was found at 25°C and 95% RH. In addition, Pal aerosols presented a larger diameter (4.7->7.0 µm) under high RH (95% RH) than under dry conditions (≤ 65% RH). These findings will play a crucial role in elucidating the influence of environmental parameters on the dynamics and transmission of Pal in aerosols. Based on our findings, preliminary recommendations for controlling airborne Pal spread involve controlling air temperature and RH, which will contribute to the effective alleviation and control of cucumber ALS disease.

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.

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.