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.

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 Fusarium oxysporum f. sp. apii race 4 Causing Fusarium Yellows on Celery in China.

Celery (Apium graveolens var. dulce), which belongs to the family Apiaceae, is one of the most widely cultivated vegetable crops in the world. During 2020 and 2021, celery plants with Fusarium yellows and root rot were observed in four approximately 0.3 ha sized fields located in Zhaili village (118°74'E, 36°67'N) of Shouguang city, Shandong province, China. Almost 50% of the plants were infected. Disease symptoms were comprised of wilting of outer-older leaves, overall stunted growth, rotted roots and stems, with eventual death of plants. A total of 7 diseased plants were collected from 4 fields and used for isolation and identification of the causal agent. Diseased root tissues were cut into 3 × 3 mm pieces from the edge of the rotting region, surface sterilized by soaking in 75% ethanol for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 28°C for 6 days in the dark. A total of 19 morphologically similar fungal isolates were obtained by single-spore subcultures. The colonies produced abundant, loosely floccose, white aerial mycelia and pale purple pigmentation on PDA. Microconidia were hyaline, zero to one septate, and ranged from 1.7 - 3.6 × 5.3 - 13.7 µm (n = 70). Macroconidia were falciform, hyaline, mostly four to five septate, and ranged from 2.2 - 4.2 × 12.4 - 45.4 µm in size (n = 70). These morphological characteristics were consistent with Fusarium oxysporum (Leslie and Summerell 2006). The genomic DNA of 19 isolates was extracted using the Plant Genomic DNA Kit (Tiangen, China). The translation elongation factor-1α (TEF-1α) and IGS rDNA regions were amplified with primers EF1/EF2 (O' Donnell et al. 1998) and iNL11/FoIGS-R (Epstein et al. 2017). BLAST analysis showed that 19 isolates were highly similar to Fusarium oxysporum, with 100% for TEF-1α (MN507109) and 99% for IGS rDNA (MT671188), respectively. The resulting 683-bp TEF-1α and 930-bp IGS rDNA sequences of isolate QC20091622 were deposited in GenBank with accession nos. ON260806 for TEF-1α and ON260805 for IGS rDNA, respectively. In a maximum-likelihood phylogenetic analysis based on TEF-1α and IGS rDNA sequences of F. oxysporum, using MEGAX software, isolate QC20091622 was grouped in the same clade with F. oxysporum f. sp. apii race 4, with a low bootstrap value of 54 between race 3 and race 4, indicating that the races are not distinguishable using only these two loci, as reported by Epstein et al (2022). Additional loci and other diagnostic methods are required to identify the race. Furthermore, the total DNA of 19 isolates was amplified by race-specific primers N4851-F/R (F. oxysporum f. sp. apii race 2) and N3875-2F/R (race 4), respectively (Epstein et al. 2017), and 187 bp product was amplified with primer pair N3875-2F/R, but none with primer pair N4851-F/R, so the isolates were identified as F. oxysporum f. sp. apii race 4. Pathogenicity of the 19 isolates was tested on potted celery plants (cv. 'Baimiao'). Ten healthy 6-week-old celery plants were inoculated by dipping the roots in a conidial suspension (107 conidia/mL) for 30 min. Control plants were dipped in sterile distilled water. The plants were then grown in a greenhouse maintained at 15°C (night)/26°C (day) and 90% relative humidity with natural daylight. The pathogenicity test was repeated twice. All inoculated plants started to wilt and developed root rot symptoms 14 days later, which were similar to those observed in the fields. The control plants remained healthy. F. oxysporum f. sp. apii race 4 was reisolated from the symptomatic roots, and their identity was confirmed by PCR, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum f. sp. apii race 4 causing root rot on celery in China. F. oxysporum f. sp. apii race 4 has been a destructive pathogen in celery, prevention and control measures should be considered.

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.

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.

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.

First Report of Fusarium solani Species Complex Causing Vascular Wilt on Cauliflower in China.

Cauliflower (Brassica oleracea var. botrytis L.), which belongs to the family Cruciferae, is a cool-season vegetable with green leaves around a large hard white head of flowers. China is the leading cauliflower and broccoli producing country in the world, with approximately 10.71 MT production (FAOSTAT 2019). During September 2018 to July 2019, wilting symptoms were observed on cauliflower in several commercial fields, with approximately 45% to 65% disease incidence in Shen county (115°48'E, 35°98N) of Liaocheng city, Shandong province, China. Plant stunting, leaves yellowing and wilting, and dark brown, hollow appearance of vascular stem tissues were the symptoms prominently observed. To isolate the causal organism, nine symptomatic tissues were collected and cut into small pieces (5 × 5 mm), disinfected in 75% ethanol for 30 s, rinsed three times in sterile water, transferred onto potato dextrose agar (PDA) medium. The plates were then incubated in air-conditioned room at 26°C with an artificial 12 h light-dark cycle provided by incandescent lamp. In total, 15 single-spore isolates were obtained and morphological characterization of 15 isolates was done on both PDA and carnation leaf agar (CLA; Leslie and Summerell 2006). The mycelia on PDA were initially white, fluffy, later becoming brown, and the underside of the colonies were light brown. Typical macroconidia were abundant on CLA. Macroconidia were hyaline, slightly curved, one to five septa, both ends were smooth, measuring 3.7 to 6.4 µm × 23.7 to 38.1 µm (n = 40). Microconidia were oval to cylindrical, hyaline, zero to one septate, measuring 2.0 to 4.1 × 4.3 to 10.3 µm (n = 40). Chlamydospores were terminal or intercalary, solitary or in pairs, globose to oval, thick wall, smooth or rough, 6.3 to 9.8 µm. Based on morphological characteristics, all of the 15 isolates were identified as Fusarium solani (Leslie and Summerell 2006). The isolates were further identified based on PCR amplification. The ITS, mtSSU, EF-1α and RPB2 genes were amplified using primers ITS1/ITS4, NSM1/NSM2 (Li et al. 1994), EF-1 Ha/EF-2Tb (O'Donnell et al. 1998) and RPB2-5F2/fRPB2-7cR (O'Donnell et al. 2008). BLAST analysis showed that 15 isolates were highly similar to F. solani species complex, with 100% similarity for ITS (AB470904.1), mtSSU (KF125009.1), EF-1α (KF372878.1), and RPB2 (MK048113.1), respectively. The sequences of isolate HYC1410080102 had been deposited in GenBank with accessions MT378292.1 for ITS, MT383122.1 for mtSSU, OK595059.1 for EF-1α and OK595060.1 for RPB2, respectively. Pathogenicity of the 15 isolates were conducted on 4-true-leaf seedlings cv. Jinsong by dipping the roots into a conidial suspension (107 conidia/mL) for 10 min. The conidial was prepared from 7-day old cultures grown on CLA at 26°C and suspended in sterilized water. Control plants were dipped in sterile distilled water. All treated seedlings were planted in 5.0 cm diameter plastic pots containing pasteurized soil matrix. Then the plants were kept in a greenhouse at 15°C (night)/26°C (day) and 80%RH with natural daylight. Twelve days later, brown lesions appeared on stem bases in all inoculated cauliflowers, and finally, the plants wilted, similar to those observed in the field. The control plants remained healthy. Re-isolation of the infected tissues showed same morphological characteristics of F. solani as the original isolates, which were verified using PCR. To our knowledge, this is the first report of F. solani causing cauliflower wilt in China and the world (Farr and Rossman 2021). F. solani is a destructive pathogen with a broad host range worldwide and is responsible for significant crop losses, prevention and control measures should be considered.

First Report of Fusarium oxysporum Causing Wilt on Coriander in China.

Coriander (Coriandrum sativum L), which belongs to the family Apiaceae, is a medicinal and aromatic plant. In China, coriander is widely cultivated in several parts as a vegetable crop. During August 2019 to June 2020, wilting symptoms were observed on coriander (cv. 'Tiegan') in a commercial plantation, with disease incidence of approximately 25 to 40% in Xiajiawang village (118°88'E, 35°46'N) of Linyi city, Shandong province, China. Symptoms included wilting and leaf yellowing, plant stunting, root rot, and vascular discoloration of the stem bases and roots. A total of eight symptomatic plants were uprooted and collected from three fields. To determine the cause of the disease, symptomatic root tissues were excised, surface disinfected with 75% ethanol for 30s, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 28°C for 6 days. In total, 10 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The morphology of multiple colonies was consistent and originally white, later becoming light to dark purple in color with abundant aerial hyphae. Macroconidia were hyaline and falcate, straight to slightly curved, 3-4 septate, 27.86 to 34.23 × 4.07 to 6.13 µm (n = 30), with apical cells curved and basal cells foot-shaped. Microconidia were hyaline, oval or ellipsoid, 0-1 septate, with a flat base, measuring 5.67 to 9.37 × 3.66 to 5.40 µm (n = 30). These morphological characteristics resembled those of Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen, China). The nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and mitochondrial small subunit (mtSSU) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1Ha/EF2Tb (O' Donnell et al. 1998) and NMS1a/NMS2b (Li et al. 1994). The resulting ITS (550-bp), TEF1-α (681-bp) and mtSSU (692-bp) sequences of isolate QC20091601 were deposited in GenBank (accession nos. MW900439, MW692008 and MW711738, respectively). BLAST analysis demonstrated 100% identities to the ITS, TEF-1α and mtSSU sequences of F. oxysporum (MN856370.1, MN507110.1 and MN386808.1), respectively. According to the morphological and molecular identification, the fungus was identified as F. oxysporum. In the pathogenicity test, healthy coriander plants (cv. 'Tiegan') at the 4-true-leaf stage were inoculated by dipping the roots into a conidial suspension of 1 × 107 conidia/mL for 10 min. Plants dipped in sterile distilled water served as controls. All treated plants were placed in a greenhouse maintained at temperature 30°C and 80% relative humidity. Ten days later, inoculated plants developed typical symptoms of leaf yellowing, wilting and vascular discoloration, which were identical to those observed in the fields, whereas the control plants remained healthy. F.oxysporum was reisolated from the symptomatic roots, and its identity was confirmed by PCR with the primes described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum as a pathogen on coriander in China. F. oxysporum is a destructive plant pathogen with an unusually broad host range and worldwide distribution, prevention and control measures should be taken in advance.

Ferroelectric and ferromagnetic properties of Pb(Ti(0.8)Fe(0.2))O(3-δ) thin film.

Single phase Pb(Ti(0.8)Fe(0.2))O(3-δ) thin films with a thickness of 210 nm and 120 nm were fabricated on Pt/Ti/SiO(2)/Si substrate by a chemical solution deposition technique. The thin film with a thickness of 210 nm showed a homogeneous microstructure, low porosity, low oxygen vacancies, and preferred orientation. It had negligible leakage current and well saturated ferroelectric hysteresis loop compared with the Pb(Ti(0.8)Fe(0.2))O(3-δ) bulk sample. Polarization fatigue characteristic indicated that this film has a potential application as a switcher in some electrical devices. The saturation magnetization in the Fe-doped PbTiO(3) film is weaker than that for bulk sample, and its ferromagnetism is correlated to the F-center exchange (FCE) mechanism. The present results revealed the multiferroic nature of the Pb(Ti(0.8)Fe(0.2))O(3-δ) thin film.