First report of Rhizopus oryzae causing rhizome rot on ginger in China.

Ginger (Zingiber officinale Rosc.) is a herbal plant, widely grown in China for its medicinal and culinary purposes. In July 2020, a new rhizome rot disease was observed on ginger in Laiwu, Shandong Province, China. The disease symptoms were observed on both above-ground and underground plant parts. The above ground stems and leaves becoming withered and yellow, and water-soaked symptoms were observed on the collar region. The diseased rhizomes were poorly developed with brown lesion and eventually they would rot, without offensive odors. Disease incidence was estimated at approximately 5% across the survey area. To isolate the pathogen, tissues from 30 rhizomes were cut from the border between diseased and healthy tissue, surface sterilized in 75% alcohol for 15 s, soaked in 0.1% mercuric chloride for 1 min, washed with sterile distilled water three times, and plated on potato dextrose agar (PDA) at 25°C for 2-3 days. Twenty nine fungal isolates with similar morphological characteristics were obtained and pure cultures were obtained using single spore isolation. The colony of AQJ-1, a representative isolate, on PDA was cottony, fluffy, white, and beige coloration on the reverse side at first, and subsequently many black sporangia were produced. The sporangia were black, sub-globose, and 45.2-181.7 µm (n = 50) in diameter. The sporangiospores were unequal, globose or sub-globose, about 3.2-8.7 × 4.6-12.3µm (n = 50) in diameter. For the molecular characterization, genomic DNA was extracted by modified CTAB method (Niu et al., 2008). Internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were amplified using the primer pairs ITS1/ITS4 (White et al., 1990) and MEF10/MEF4 (Abe et al., 2007), respectively. The ITS and EF-1α sequences of isolate AQJ-1 were submitted to GenBank (MN606288 and MN735220, respectively). The BLASTn analysis of the sequences showed 99%-100% similarity to the sequences of R. oryzae strain CBS 120.12 (MH854609, AB281529, respectively). Therefore, based on morphological and molecular characteristics, isolate AQJ-1 was identified as R. oryzae. For pathogenicity tests, thirty ginger seedlings (Laiwu Big Ginger) were grown for 30 days in plastic pots and removed from the pots and the rhizomes washed in running tap water. The rhizomes of fifteen ginger seedlings were attached to a 7 mm agar disk from a plate containing 2-day-old mycelium, and the other fifteen seedlings were attached to agar disk without mycelium as control. Then the inoculated and control seedlings were planted in pots and were kept in separate chambers in a greenhouse at 25±2 °C. After 14 days, the same symptoms of rhizome rot were observed in all inoculated plants as previously described, and no symptoms were observed on the control plants. The pathogen was re-isolated from symptomatic tissues, and was identified as R. oryzae, which full-filled the Koch's postulates. To our knowledge, this is the first report of R. oryzae causing rhizome rot on ginger in China. This disease may pose a potential threat to ginger production in China.

First report of Cladosporium tenuissimum causing leaf spot of Arachis hypogaea in China.

Peanut (Arachis hypogaea L.), one of the most important oilseed crops in tropical and subtropical regions of the world (Kumar and Kirti 2011), is widely cultivated for its high protein and oil content in seeds. In August 2019, about 30% of A. hypogaea plants were found infected by leaf spot in the peanut-growing regions of Shandong Province, China. Disease symptoms appeared as the irregular and brown necrotic lesions on leaves that were 0.5 to 5.0 mm in diam. Twenty symptomatic plants were randomly sampled from peanut planting areas in Weihai and Yantai City. Small pieces (3 mm2) were cut from lesions, dipped in a 0.5% NaClO for 10 min, rinsed three times with sterilized distilled water, dried, placed onto potato-dextrose agar (PDA), and incubated in the dark at 25°C for 10 days. Three typical Cladosporium-like strains were isolated from diseased leaves of peanut. The colonies were grey to olivaceous green, reverse olivaceous black and woolly. The conidiophores were solitary, macronematous, unbranched or branched, straight or flexuous, cylindrical, slightly swollen at the apex, smooth. Conidiogenous cells were integrated, terminal and intercalary, with numerous loci on nodulose swelling. Ramoconidia were cylindrical, oblong, fusiform, 8.0 to 19.5×2.0 to 4.5 µm, aseptate or 1 septum, pale brown. Conidia were catenate, in densely branched chains, ellipsoid, ovoid, limoniform, aseptate, 4.0 to 11.5×2.5 to 5.5 µm, smooth, with conspicuous hila. The conidia easily break off from the chains. The morphological characteristics of these isolates matched the descriptions of Cladosporium tenuissimum (Bensch et al. 2010). For the molecular identification, the partial actin (act) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the respective primers ACT-512F/ACT-783R and EF1-728F/EF1-986R (Carbone and Kohn 1999). The representative sequences, deposited in GenBank (act: OL332701, OL332702 and OL332703; tef1: OL322090, OL322091 and OL322092), exhibited 99.6% and 100% identical to C. tenuissimum ex-type isolate CBS 125995 (HM148687 and HM148442). Phylogenetic analysis was done by Neighbor-Joining (NJ) analysis based on act+tef1 sequences. These three isolates were identified as C. tenuissimum by morphological and molecular characteristics. Pathogenicity of each C. tenuissimum isolate was tested on peanut in the greenhouse at 28°C with 75% relative humidity. Twenty plants of A. hypogaea were inoculated with the conidial suspension (1.0 × 105 conidia/ml) on the leaf surface. Ten plants were mock inoculated with sterile water as controls. Within 2 weeks, inoculated plants exhibited dark necrotic lesions on leaves which were similar to the symptoms observed in the field, while the mock inoculated plants remained symptomless. The fungal pathogen which was reisolated from inoculated rather than mock inoculated leaf tissues was identical to the original pathogen on the basis of morphological and molecular analysis, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. tenuissimum on peanut in China. The C. tenuissimum infection poses a serious threat by reducing the yield and quality of peanut in Shandong Province. This research is especially valuable to enhance epidemiological studies and implement effective control strategies.

Background-dependent Wolbachia-mediated insecticide resistance in Laodelphax striatellus.

Although facultative endosymbionts are now known to protect insect hosts against pathogens and parasitoids, the effects of endosymbionts on insecticide resistance are still unclear. Here we show that Wolbachia are associated with increased resistance to the commonly used insecticide, buprofezin, in the small brown planthopper (Laodelphax striatellus) in some genetic backgrounds while having no effect in other backgrounds. In three Wolbachia-infected lines from experimental buprofezin-resistant strains and one line from a buprofezin-susceptible line established from Chuxiong, Yunnan province, China, susceptibility to buprofezin increased after removal of Wolbachia. An increase in susceptibility was also evident in a Wolbachia-infected line established from a field population in Rugao, Jiangsu province. However, no increase was evident in two field populations from Nanjing and Fengxian, Jiangsu province, China. When Wolbachia was introgressed into different genetic backgrounds, followed by Wolbachia removal, the data pointed to Wolbachia effects that depend on the nuclear background as well as on the Wolbachia strain. However, there was no relationship between Wolbachia density and the component of buprofezin resistance associated with the symbiont. The results suggest that Wolbachia effects associated with chemical resistance are complex and unpredictable, but also that they can be substantial.

Population Dynamics of Wolbachia in Laodelphax striatellus (Fallén) Under Successive Stress of Antibiotics.

Wolbachia are the most common symbionts in arthropods; antibiotic treatment for eliminating the symbionts from their host is necessary to investigate the functions. Tetracycline antibiotics are widely used to remove endosymbiont Wolbachia from insect hosts. However, very little has been known on the effects of tetracycline on population size of Wolbachia in small brown planthopper (SBPH), Laodelphax striatellus (Fallén), an important insect pest of rice in Asia. Here, we investigated the dynamics of Wolbachia population density in females and males of L. striatellus by real-time fluorescent quantitative PCR method. The Wolbachia density in females and males of L. striatellus all declined sharply after treatment with 2 mg/mL tetracycline for one generation, and continued to decrease to a level which could not be detected by both qPCR and diagnostic PCR after treated for another generation, then maintained at 0 in the following three generations with continuous antibiotic treatment. Wolbachia infection did not recover in L. striatellus after stopping tetracycline treatment for ten generations. This is the first report to precisely monitor the population dynamics of Wolbachia in L. striatellus during successive tetracycline treatment and after that. The results provide a useful method for evaluating the efficiency of artificial operation of endosymbionts.

Variations in Endosymbiont Infection Between Buprofezin-Resistant and Susceptible Strains of Laodelphax striatellus (Fallén).

The endosymbionts Wolbachia and Rickettsia have been shown to be correlated with the insecticide resistance of mosquito and whitefly. The small brown planthopper (SBPH), Laodelphax striatellus, harbours many species of endosymbionts, and has developed a high resistance to buprofezin in China. In this study, we examined the species and the infection incidences of endosymbionts in a buprofezin-resistant (BR) strain, a buprofezin-susceptible (BS) strain, and the BR strain after exposure to buprofezin, and we also investigated the change in buprofezin susceptibility after removal of Wolbachia from the BR strain. The results showed that Wolbachia infection incidences were 100% in both the BR and BS strains, but the Wolbachia density in the BR strain was significantly higher than that in the BS strain. There were no significant differences in Arsenophonus infection incidence between the two strains. However, the infection incidence of Serratia and double infection incidence of Serratia + Wolbachia in the BR strain were significantly higher than that in the BS strain. After the BR strain was exposed to 1200 mg/L buprofezin, the infection incidence of Arsenophonus in the surviving individuals increased, and the infection rate of Serratia did not differ, but the double infection incidence of Serratia + Wolbachia decreased. And when a Wolbachia-infected line originating from the BR strain was cleared of Wolbachia, its susceptibility to buprofezin increased. The results suggest that Serratia and Wolbachia infection might improve the buprofezin resistance of SBPH.

Surgical prediction of neonatal necrotizing enterocolitis based on radiomics and clinical information.

PURPOSE: To assess the predictive value of radiomics for surgical decision-making in neonatal necrotizing enterocolitis (NEC) when abdominal radiographs (ARs) do not suggest an absolute surgical indication for free pneumoperitoneum. METHODS: In this retrospective study, we finally included 171 newborns with NEC and obtained their ARs and clinical data. The dataset was randomly divided into a training set (70%) and a test set (30%). We developed machine learning models for predicting surgical treatment using clinical features and radiomic features, respectively, and combined these features to build joint models. We assessed predictive performance of the different models by receiver operating characteristic curve (ROC) analysis and compared area under curve (AUC) using the Delong test. Decision curve analysis (DCA) was used to assess the potential clinical benefit of the models to patients. RESULTS: There was no significant difference in AUC between the clinical model and the four radiomic models (P > 0.05). The XGBoost joint model had better predictive efficacy and stability (AUC, training set: 0.988, test set: 0.959). Its AUC in the test set was significantly higher than that of the clinical model (P < 0.05). DCA showed that the XGBoost joint model achieved higher net clinical benefit compared to the clinical model in the threshold probability range (0.2-0.6). CONCLUSION: Radiomic features based on AR are objective and reproducible. The joint model combining radiomic features and clinical signs has good surgical predictive efficacy and may be an important method to help primary neonatal surgeons assess the surgical risk of NEC neonates.

Does Resistance to Buprofezin Improve Heat and Cold Tolerance of Laodelphax striatellus (Hemiptera: Delphacidae)?

There is ample evidence that insecticide resistance causes fitness costs and benefits in pests, while the impact of insecticide resistance on thermotolerance of pests is mostly unclear. The Laodelphax striatellus (Fallén), is an important rice insect pest, which has developed resistance to buprofezin in China. Here, we investigated differences in heat tolerance and cold tolerance among L. striatellus lines with variable buprofezin resistance. The lethal time for 50% of the individuals to die (LT50) at 40 °C increased with an increase in buprofezin resistance level, whereas both the survival rate under -22 °C and the supercooling point of planthoppers did not differ significantly between resistant and susceptible strains. The metabolic enzyme carboxylesterase was found to have an association with buprofezin resistance. Our research showed that buprofezin resistance was positively related with heat tolerance in L. striatellus, but it had no effect on cold tolerance. Insecticide resistance in L. striatellus may therefore have broader implications for the ecology of L. striatellus, and the management of buprofezin resistance in this pest may be challenging.