Identification and characterization of pathogens causing saffron corm rot in China.

Corm rot is the most important disease of saffron, for which fungi from several genus such as Fusarium spp. Penicillium spp. and Botrytis spp., have been previously reported to be the pathogens. In this research, we used a combination of amplicon sequencing and traditional isolation methods to identify the causal agents, main infection source. The diversity of microbial communities in diseased saffron corms and soil decreased significantly compared with healthy corms and soil. The contents of Penicillium and Botrytis in healthy and diseased corms were similarly high, indicating that them were not directly related to the occurrence of corm rot. But the relative abundance of Fusarium, Cadophora and Fusicolla were significantly higher in the diseased corms than healthy ones. The abundance of Fusarium increased, while the abundance of Oidiodendron, Paraphaeosphaeria and the endophytic beneficial bacteria Pseudomonas decreased, which may relate to the occurrence of the disease. The co-occurrence network diagram showed that the correlation between fungal and bacterial communities was mainly positive. Plant pathogens were relatively abundant in the diseased soil, according to functional gene prediction. At the same time, we also collected 100 diseased corms from the fields in Jiande, where is known as the "hometown of saffron." All isolated pathogenic strains were identified as Fusarium oxysporum through morphological observation and phylogenetic tree analysis of ITS, Tef-1α and ß-tubulin. To better clarify the biological characteristics of F. oxysporum, we cultured the isolates at different temperatures and pH values. The optimum temperature for mycelial growth and sporulation was 25°C, pH 6，carbon sources sorbitol and nitrogen sources, peptone. In short, our results suggests that F. oxysporum was the pathogen causing corm rot in Jiande and corms other than soils are the main primary infection source. These new understanding of saffron corm rot will provide the theoretical basis for its better and efficiently management.

Advancements in the Use of Bacteriophages to Combat the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

Over the last several decades, kiwifruit production has been severely damaged by the bacterial plant pathogen Pseudomonas syringae pv. actinidiae (Psa), resulting in severe economic losses worldwide. Currently, copper bactericides and antibiotics are the main tools used to control this bacterial disease. However, their use is becoming increasingly ineffective due to the emergence of antibiotic resistance. In addition, environmental issues and the changes in the composition of soil bacterial communities are also concerning when using these substances. Although biocontrol methods have shown promising antibacterial effects on Psa infection under in vitro conditions, the efficiency of antagonistic bacteria and fungi when deployed under field conditions remains unclear. Therefore, it is crucial to develop a phage-based biocontrol strategy for this bacterial pathogen. Due to the specificity of the target bacteria and for the benefit of the environment, bacteriophages (phages) have been widely regarded as promising biological agents to control plant, animal, and human bacterial diseases. An increasing number of studies focus on the use of phages for the control of plant diseases, including the kiwifruit bacterial canker. In this review, we first introduce the characteristics of the Psa-induced kiwifruit canker, followed by a description of the diversity and virulence of Psa strains. The main focus of the review is the description of recent advances in the isolation of Psa phages and their characterization, including morphology, host range, lytic activity, genome characterization, and lysis mechanism, but we also describe the biocontrol strategies together with potential challenges introduced by abiotic factors, such as high temperature, extreme pH, and UV irradiation in kiwifruit orchards. The information presented in this review highlights the potential role of phages in controlling Psa infection to ensure plant protection.

Pseudomonas bijieensis Strain XL17 within the P. corrugata Subgroup Producing 2,4-Diacetylphloroglucinol and Lipopeptides Controls Bacterial Canker and Gray Mold Pathogens of Kiwifruit.

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) and gray mold caused by Botrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa and B. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa and B. cinerea, Psa-associated leaf necrosis, and B. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa and B. cinerea. Genome-based taxonomy revealed that strain XL17 belongs to Pseudomonas bijieensis within the P. corrugata subgroup of the P. fluorescens species complex. Among the P. corrugata subgroup containing 31 genomospecies, the presence of the phl operon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified that P. bijieensis XL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit.

First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China.

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05', E119°38'), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 µm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 µm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.

Evaluation of joint effects of cyprodinil and kresoxim-methyl on zebrafish, Danio rerio.

Aquatic organisms are usually exposed to a mixture of pesticides instead of individual chemicals. However, risk assessment of pesticides is traditionally based on toxicity data of individual compounds. In this study, we aimed to examine the joint toxicity of two fungicides cyprodinil (CYP) and kresoxim-methyl (KRM) to zebrafish (Danio rerio) using a systematic experimental approach. Results from 96-h semi-static test indicated that the LC50 values of KRM to D. rerio at multiple life stages (embryonic, larval, juvenile and adult stages) ranged from 0.034 (0.015-0.073) to 0.61 (0.39-0.83) mg a.i. L-1, which were higher than those of CYP ranging from 1.05 (0.88-1.52) to 4.42 (3.24-6.02) mg a.i. L-1. Pesticide mixtures of CYP and KRM exhibited synergistic effect on embryonic zebrafish. The activities of carboxylesterase (CarE) and cytochrome P450 (Cyp450) were significantly altered in most of the individual and combined exposures compared with the control group. The expressions of seven genes (Mnsod, cyp17, crhr 2, crh, gnrhr 4, gnrhr 1 and hmgrb) were significantly altered upon exposure to combined pesticides compared with their individual pesticides. Collectively, these findings suggested joint effects should be considered in the risk assessment of pesticides and development of water quality criteria for the protection of aquatic environment.

Combined toxicity of imidacloprid and three insecticides to the earthworm, Eisenia fetida (Annelida, Oligochaeta).

Although the earthworm Eisenia fetida has been used in many ecotoxicological studies in recent years, most of these studies have only focused on assessing the effects of individual insecticides. In the present study, we aimed to compare the individual and combined toxic effects of imidacloprid and three insecticides (phoxim, chlorpyrifos, and lambda-cyhalothrin) on E. fetida. We showed that imidacloprid had the highest intrinsic toxicity to the worms in filter paper contact test, followed by phoxim and lambda-cyhalothrin, while the least toxicity was found from chlorpyrifos. Moreover, 14-day soil toxicity test revealed that the highest toxicity was still detected for imidacloprid with an LC50 value of 2.82 (2.61∼3.17) mg a.i. kg-1 dry weight (DW), followed by chlorpyrifos with an LC50 value of 384.9 (353.5∼440.3) mg a.i. kg-1 DW. Meanwhile, a relatively less toxicity was found for lambda-cyhalothrin with an LC50 value of 560.3 (475.9∼718.5) mg a.i. kg-1 DW, while the lowest toxicity to E. fetida was observed for phoxim with an LC50 value of 901.5 (821.3∼1017) mg a.i. kg-1 DW. In addition, significant synergistic responses were found from the ternary mixture of imidacloprid-phoxim-lambda-cyhalothrin and quaternary mixture of imidacloprid-phoxim-chlorpyrifos-lambda-cyhalothrin in both bioassay systems. Therefore, our findings highlighted that the simultaneous presence of several insecticides in the soil environment might lead to increased toxicity, resulting in serious damage to the nontarget organisms compared with individual insecticides.

Individual and mixture effects of five agricultural pesticides on zebrafish (Danio rerio) larvae.

In the present study, we evaluated the individual and mixture toxicities of imidacloprid and other four pesticides (atrazine, chlorpyrifos, butachlor, and λ-cyhalothrin) to the zebrafish (Danio rerio) larvae in order to clarify the interactive effects of pesticides on aquatic organisms. Results from the 96-h semi-static toxicity test indicated that chlorpyrifos, λ-cyhalothrin, and butachlor had the highest toxicities to D. rerio with an LC50 value ranging from 0.28 (0.13∼0.38) to 0.45 (0.31∼0.59) mg AI L-1, followed by atrazine with an LC50 value of 15.63 (10.71∼25.76) mg AI L-1, while imidacloprid exhibited the least toxicity to the organisms with an LC50 value of 143.7 (99.98∼221.6) mg AI L-1. Seven pesticide mixtures (two binary mixtures of imidacloprid + atrazine and imidacloprid + λ-cyhalothrin, two ternary mixtures of imidacloprid + atrazine + λ-cyhalothrin and imidacloprid + butachlor + λ-cyhalothrin, two quaternary mixtures of imidacloprid + atrazine + chlorpyrifos + λ-cyhalothrin and imidacloprid + chlorpyrifos + butachlor + λ-cyhalothrin, and one quinquenary mixture of imidacloprid + atrazine + chlorpyrifos + butachlor + λ-cyhalothrin) exhibited synergistic effects with equitoxic ratio and equivalent concentration on the zebrafish. Our results highlighted that the simultaneous presence of several pesticides in the aquatic environment might lead to increased toxicity, causing serious damage to the aquatic ecosystems compared with their individual toxicities. Therefore, the toxic effects of both individual pesticides and their mixtures should be incorporated into the environmental risk evaluation of pesticides.

Buprofezin susceptibility survey, resistance selection and preliminary determination of the resistance mechanism in Nilaparvata lugens (Homoptera: Delphacidae).

BACKGROUND: Buprofezin has been used for many years to control Nilaparvata lugens (Stål). Assessment of susceptibility change in the insect is essential for maintaining control efficiency and resistance management. RESULTS: Eleven-year surveys showed that most field populations were susceptible before 2004. However, substantially higher levels of resistance (up to 28-fold) were found in most of the rice fields in China after 2004. A field population was collected and periodically selected for buprofezin resistance in the laboratory. After 65 generations (56 were selected), the colony successfully obtained 3599-fold resistance to buprofezin. Synergism tests showed that O,O-diethyl-O-phenyl phosphorothioate (SV1), piperonyl butoxide (PBO) and diethyl maleate (DEM) increased buprofezin toxicity in the resistant strain by only 1.5-1.6 fold, suggesting that esterases, P450-monooxygenases and glutathione S-transferases had no substantial effect on buprofezin resistance development. CONCLUSION: The results from this study indicate that N. lugens has the potential to develop high resistance to buprofezin. A resistance management program with rotation of buprofezin and other pesticides may efficiently delay or slow down resistance development in the insect. Further investigation is also necessary to understand the resistance mechanisms in N. lugens.