Stem Lesions Research Articles

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Cerebrovascular Diseases and Autonomic Failure

Cerebral vessels are innervated by sympathetic and parasympathetic nerves, as well as the trigeminal nerve. Under physiological conditions, neural regulation contributes to coupling of cerebral blood flow (CBF) and metabolism and autoregulation of CBF against systemic blood pressure. It also plays a key role in vasodilatation distal to narrowed vascular lesions associated with atherosclerosis, hyperperfusion syndrome that occurs after vascular reconstruction in patients with chronic ischemic diseases, reversible cerebral vasoconstriction syndrome, and migraine. Moreover, cerebrovascular disease can cause autonomic dysfunction; frontal lobe and brain stem lesions are known to result in urinary incontinence, mydriasis, and Horner's syndrome.

First Report of Sclerotinia Fruit and Crown Rot Caused by Sclerotinia sclerotiorum on Short-Day Strawberry in Maryland.

With over 1,000 producers in the Mid-Atlantic region, short-day strawberry (Fragaria x Ananassa Duch.) represents an economically important industry. In spring of 2016, a white rot was observed on strawberry stem, crown, and fruit tissue at two Maryland farms. Light to dark brown necrotic lesions, white mycelium and medium to large black sclerotia were present on petioles and fruit (Fig. 1). These symptoms were most consistent with Sclerotinia fruit rot and crown rot caused by Sclerotinia sclerotiorum (Lib.) de Bary (Maas 1998), not previously described in the region (Farr and Rossman 2019). Lesion margins were excised, surface disinfested (70% EtOH for 30 s and 0.1% NaClO for 1 min) and placed onto potato dextrose agar (PDA) amended with 0.03% tetracycline. Single hyphal tip cultures were obtained which formed fluffy white to tan mycelium, no asexual conidia, and numerous pigmented sclerotia resembling S. sclerotiorum (Bolton 2006). DNA was extracted from five isolates and the internal transcribed spacer (ITS) region was amplified by PCR using primers ITS1/ITS4; all five ITS sequences (533 bp) had 99-100% similarity with S. sclerotiorum (GQ375746) in Genbank. Sequences of SL1380 and SL1382 were submitted to NCBI under the accession numbers MK429976 and MK429977, respectively. These two isolates were evaluated for abilities to cause fruit and stem petiole rot. Pathogenicity on fruit was examined by placing a 5 mm plug from five-day-old cultures on surface disinfested fruits, with mock-inoculated (PDA plug only) and non-inoculated controls. Fruit were placed in incubators (~98% RH, 22°C, 10:14 hrs L:D) and arranged in a complete block design with three blocks and five fruit/treatment/block; the study was conducted twice. White hyphae and sclerotia formed on 41 to 53% of fruit inoculated with both isolates, necrotizing fruit within 6 days post inoculation (DPI); Sclerotinia fruit rot did not develop in controls. Pathogenicity on petioles was examined in a replicated study on three-month-old cv. Flavorfest using a petiole inoculation method described in (Peltier and Grau 2008); mock-inoculated stems were placed in sterile PDA. Plants were arranged in the greenhouse (~20-28°C; 12:12 hrs L:D) in a randomized complete block design with three blocks and three plants/treatment. Dark brown necrosis averaging 5.8 ± 0.2 mm in length developed on 100% of plants in both isolate treatments within 3 DPI and by 6 DPI had expanded to 42.1 ± 4.8 mm on average, spreading into the crown on some plants. Necrosis did not extend beyond 2 mm from the cut tip in controls. The pathogen was recovered from surface disinfested tissue for both isolates. In a subsequent study, stem rot susceptibility was compared among four short-day strawberry cultivars common to the region, cvs. Flavorfest, Chandler, Allstar and Honeoye (same method as above). Stem lesions were 2 to 3 times longer on cv. Flavorfest (41.78(ave) ± 6.73(se) mm) compared to all other cultivars (13.68(ave) ± 1.56(se) mm) (P < 0.0001, Tukey's test). To our knowledge, this is the first report of both Sclerotinia fruit and crown rot on strawberries anywhere in the mid-Atlantic region. Our results indicate that cv. Flavorfest, a recently developed cultivar with expanding acreage, may be more susceptible to Sclerotinia. These are both unusual diseases circumglobally, especially Sclerotinia fruit rot (Farr and Rosman 2019, Maas 1998); the new occurrence in this region may be associated with increased use of this cultivar.

Acupuncture Reduces the Risk of Dysphagia in Stroke Patients: A Propensity Score-Matched Cohort Study.

Background: Post-stroke dysphagia (PSD) affects the quality of life in stroke patients, impairs their rehabilitation ability, and causes other complications following stroke. Currently, there is currently some understanding of PSD risk factors, but its protective factors remain largely unknown.Objective: To analyze the effects of acupuncture (AP) on dysphagia in stroke patients and explore its potential as a preventive therapy.Methods: Patients with a diagnosis of stroke from 2010 to 2019 were selected and followed until 2020, utilizing factors such as age, gender, stroke location, stroke type, and baseline comorbidity. To compare the incidence of dysphagia, equal numbers of stroke patients treated with and without AP (n = 1,809) were matched by 1:1 propensity scoring. The Cox proportional hazards model and Kaplan-Meier method were used to assess the risk of dysphagia as an outcome measure.Results: The stroke patients treated with AP had a lower risk of dysphagia after adjusting for age, gender, stroke location, stroke type, and baseline comorbidity [adjusted hazard ratio (AHR) = 0.43, 95% confidence interval = 0.37–0.49] compared with those in the non-AP cohort. AP also decreased the risk of PSD among different gender groups. The risk ratios were AHR = 0.45 and AHR = 0.33 for males and females, respectively. AP also reduced the risk for PSD among different age groups. The risk ratios were AHR = 0.20, AHR = 0.37, AHR = 0.41, and AHR = 0.45 for the 18–39, 40–59, 60–79, and >80 years-old groups. Regarding stroke types (ischemic, hemorrhagic, and mixed type), patients treated with AP had a lower risk (AHR = 0.47, 0.28 and 0.17, respectively). With respect to stroke location, the risk of PSD in AP-treated patients was decreased regardless of location: brain stem (AHR = 0.41), diencephalon (AHR = 0.13), or multiple lesions (AHR = 0.40), the risk of PSD in AP-treated patients was decreased. For all baseline comorbidities, AP attenuated the risk of dysphagia. The cumulative incidence of dysphagia was remarkably lower in the AP group than in the non-AP group (log-rank test, P = 0.000).Limitations: First, this was a single-center clinical retrospective study. Second, we did not classify the severity of stroke and dysphagia. Third, all data were extracted manually. Lastly, the sample size was relatively small. Thus, future studies with larger sample sizes are warranted to verify our findings.Conclusion: Acupuncture treatment attenuates the risk of dysphagia in stroke patients. Future research should increase the sample size and elaborate further on the details of the AP protocol.

Improved YOLOv5 Model for Rice Stem and Leaf Lesion Image Annotation

Improved YOLOv5 Model for Rice Stem and Leaf Lesion Image Annotation

Root Plate Growth in Sunflower and Its Relevance to Sclerotinia Basal Stalk Rot

Sclerotinia basal stalk rot (BSR) of sunflower (Helianthus annuus L.) is a fungal disease of the roots that causes symptoms of wilt and a basal stem lesion. Evaluating root plate growth could improve our understanding of BSR. Separate studies were conducted to determine the effect of sunflower growth stage or genotype on root plate diameter in North American environments. Root plate diameter of cultivated hybrids at reproductive growth stages was 3 to 4 times larger than vegetative stages. Cultivated hybrids had larger root plate diameter than interspecific lines. These results have implications for artificial inoculation methods that evaluate genotypes for BSR resistance in the field or greenhouse. Disease escapes can occur if field-grown plants are inoculated too far from the root plate and/or too early at vegetative growth stages. Side-dressing mycelium-infested cereal grain closer (i.e., 10 cm) to plants at reproductive growth stages (i.e., R1 - R4) can increase disease pressure and reduce disease escapes. These guidelines for the field can be used to validate results from artificial inoculations in the greenhouse.

Quantitative Inheritance of Sclerotinia Stem Rot Resistance in Brassica napus and Relationship to Cotyledon and Leaf Resistances

Sclerotinia sclerotiorum is a necrotrophic fungus causing devastating stem rot and associated yield losses of canola/rapeseed (Brassica napus) worldwide, including in Australia. Developing host resistance against Sclerotinia stem rot is critical if this disease in canola/rapeseed is to be successfully managed, as cultural or chemical control options provide only partial or sporadic control. Three B. napus breeding populations, C2, C5 and C6, including the parents, F1, F2, BC1P1, and BC2P2, were used in a field study with an objective of exploring the inheritance pattern of disease resistance (based on stem lesion length [SLL]) and the genetic relationships of disease with stem diameter (SD) or days to first flowering (DTF), and to compare these new adult plant stem resistances against S. sclerotiorum with those of seedling (cotyledon and leaf) resistances in earlier studies. Heritability (broad sense) of SLL was 0.57 and 0.73 for population C2 at 3 and 5 weeks postinoculation and 0.21 for population C5 at 5weeks postinoculation. Additive genetic variance was evident within all 3 populations for DTF but not for SD. Narrow-sense heritability for DTF was 0.48 (C2), 0.42 (C5), and 0.32 (C6). SD, DTF, and SLL were all inherited independently, with no significant genetic covariance between traits in bivariate analysis. Genetic variance for SLL in populations C2 and C5 was entirely nonadditive, and there was significant nonadditive genetic covariance of SLL at 3 and 5 weeks postinoculation. Generation means analysis in population C2 supported the conclusion that complex epistatic interactions controlled SLL. Several C2 and C5 progeny showed high adult plant stem resistance, which may be critical in developing enhanced stem resistance in canola/rapeseed. Although population C6 showed no genetic variation for SLL resistance in this study, it showed significant nonadditive genetic variance at the cotyledon and leaf stages in earlier studies. We conclude that host resistance varies across different plant growth stages, and breeding must be targeted for resistance at each growth stage. In populations C2, C5, and C6, resistance to S. sclerotiorum in stem, leaf, and cotyledon was always controlled by nonadditive effects such as complex epistasis or dominance. Overall, our findings in relation to the quantitative inheritance of Sclerotinia stem rot resistance, together with the new high-level resistances identified, will enable breeders to select/develop genotypes with enhanced resistances to S.sclerotiorum.

First report of Pectobacterium versatile causing blackleg of potato in Serbia.

Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20-25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates' ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as 'Candidatus Pectobacterium maceratum' (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.

Early Blood Glucose Level Post-Admission Correlates with the Outcomes and Oxidative Stress in Neonatal Hypoxic-Ischemic Encephalopathy.

The antioxidant defense system is involved in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). To analyze the relationship between first serum blood glucose levels and outcomes in neonatal HIE, seventy-four patients were divided, based on the first glucose level, into group 1 (>0 mg/dL and <60 mg/dL, n =11), group 2 (≥60 mg/dL and <150 mg/dL, n = 49), and group 3 (≥150 mg/dL, n = 14). Abnormal glucose levels had poor outcomes among three groups in terms of the clinical stage (p = 0.001), brain parenchymal lesion (p = 0.004), and neurodevelopmental outcomes (p = 0.029). Hearing impairment was more common in group 3 than in group 1 (p = 0.062) and group 2 (p = 0.010). The MRI findings of group 3 exhibited more thalamus and basal ganglion lesions than those of group 1 (p = 0.012). The glucose level was significantly correlated with clinical staging (p < 0.001), parenchymal brain lesions (p = 0.044), hearing impairment (p = 0.003), and neurodevelopmental outcomes (p = 0.005) by Pearson’s test. The first blood glucose level in neonatal HIE is an important biomarker for clinical staging, MRI findings, as well as hearing and neurodevelopment outcomes. Hyperglycemic patients had a higher odds ratio for thalamus, basal ganglia, and brain stem lesions than hypoglycemic patients with white matter and focal ischemic injury. Hyperglycemia can be due to prolonged or intermittent hypoxia and can be associated with poor outcomes.

Brenneria goodwinii growth in vitro is improved by competitive interactions with other bacterial species associated with Acute Oak Decline.

Brenneria goodwinii, Rahnella victoriana and Gibbsiella quercinecans are three bacterial species frequently isolated together from oak displaying symptoms of Acute Oak Decline (AOD), which include weeping patches on trunks. All three bacterial species play a role in lesion formation in the current episode of AOD in Britain, although B. goodwinii is the most dominant. The ongoing research into stem lesion formation characteristic of this polybacterial syndrome has been focussed primarily on the pathogenicity, identification and taxonomy of these bacteria. As all three species were newly classified within the past ten years, there are many unanswered questions regarding their ecology and interactions with each other. To determine the effect of bacterial interactions on fitness in vitro, we examined pairwise (diculture) and multispecies (triculture) interactions between B. goodwinii, R. victoriana and G. quercinecans in oak leaf media microcosms. Additionally, the effect of co-culturing on the evolution of these species was determined and the evolved B. goodwinii strains were examined further by whole genome sequencing. Our results indicate that B. goodwinii thrived in monoculture with significantly higher viable cell counts than the other two species. Additionally, B. goodwinii performed well in pairwise culture with mutually competitive interactions observed between B. goodwinii and R. victoriana, and between B. goodwinii and G. quercinecans. In the multispecies triculture, B. goodwinii and R. victoriana appeared to exhibit co-ordinated behaviour to outcompete G. quercinecans. After four weeks B. goodwinii grown in co-culture with the other two species developed greater evolved fitness than the strain grown in monoculture as reflected by the increased viable cell counts. The competitive interactions taking place between the threes species indicated evolving improved fitness of B. goodwinii in vitro, that gave it a growth advantage over both R. victoriana and G. quercinecans which showed no significant changes in fitness. Overall, B. goodwinii gains greater benefit in terms of fitness from in vitro competitive interaction with the other two species.

Evidence for the Widespread Occurrence of Bacteria Implicated in Acute Oak Decline from Incidental Genetic Sampling

Acute Oak Decline (AOD) is complex syndrome affecting Britain’s keystone native oak species, (Quercus robur L. and Q. petraea L. (Matt.) Liebl.), in some cases causing mortality within five years of symptom development. The most distinguishable symptom is weeping stem lesions, from which four species of bacteria have been isolated: Brenneria goodwinii, Gibbsiella quercinecans, Lonsdalea britannica and Rahnella victoriana. We do not yet know where else these bacteria exist, and little is known about the relationship of the wider oak leaf microbiome (phyllosphere) to acute oak decline. Here we investigate whether incidental evidence from a large oak genome re-sequencing dataset could be used to detect these bacteria in oak foliage, and whether bacterial incidence co-varied with AOD status or location. Oak leaves and buds were sampled from 421 trees at five sites in England. Whole genomic DNA from these samples was shot-gun sequenced with short reads. Non-oak reads were extracted from these data and queried to microbial databases. Reads uniquely matching AOD-associated bacterial genomes were found to be present on trees from all five sites and included trees with active lesions, trees with historic lesions and trees without AOD symptoms. The abundance of the AOD-associated bacteria did not differ between tree health categories but did differ among sites. We conclude that the AOD-associated bacteria may be members of the normal oak microbiome, whose presence on a tree is not sufficient to cause AOD symptoms.

Genome-wide association mapping and genomic prediction for adult stage sclerotinia stem rot resistance in Brassica napus (L) under field environments

Sclerotinia stem rot (SSR) is a fungal disease of rapeseed/canola that causes significant seed yield losses and reduces its oil content and quality. In the present study, the reaction of 187 diverse canola genotypes to SSR was characterized at full flowering stage using the agar plug to stem inoculation method in four environments. Genome-wide association study (GWAS) using three different algorithms identified 133 significant SNPs corresponding with 123 loci for disease traits like stem lesion length (LL), lesion width (LW), and plant mortality at 14 (PM_14D) and 21 (PM_21D) days. The explained phenotypic variation of these SNPs ranged from 3.6 to 12.1%. Nineteen significant SNPs were detected in two or more environments, disease traits with at least two GWAS algorithms. The strong correlations observed between LL and other three disease traits evaluated, suggest they could be used as proxies for SSR resistance phenotyping. Sixty-nine candidate genes associated with disease resistance mechanisms were identified. Genomic prediction (GP) analysis with all the four traits employing genome-wide markers resulted in 0.41–0.64 predictive ability depending on the model specifications. The highest predictive ability for PM_21D with three models was about 0.64. From our study, the identified resistant genotypes and stable significant SNP markers will serve as a valuable resource for future SSR resistance breeding. Our study also suggests that genomic selection holds promise for accelerating canola breeding progress by enabling breeders to select SSR resistance genotypes at the early stage by reducing the need to phenotype large numbers of genotypes.

First Report of Epicoccum sorghinum Causing Leaf Sheath and Leaf Spot on Maize in China.

In November 2020, leaf sheath on maize (Zea mays) was detected in southeastern Jiangsu (Nantong municipality; 120.54° E, 31.58° N) in China. Physiologically mature plants, 13 weeks of cultivation (at the harvest stage), exhibited red-brown lesions in stem and leaves, and dried-up stem (Figure 1). The symptoms were observed on approximately 95% of the maize plants in a 0.8 ha maize field surrounded by old sorghum fields and the crop yield was decreased by 70-85% with respect previous years, when no disease symptoms were detected. Small pieces, approximately 0.3 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated (one isolate was obtained) and cultured on PDA medium, containing chloramphenicol (50 µg/mL), under darkness at 26 ºC for 3 days. Amplification of internal transcribed spacer (ITS), large subunit (LSU), actin (ACT) and β-tubulin (TUB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R and T1/Bt2b primers, respectively (Ma et al. 2021). Sequences were submitted to GenBank under accession numbers MW800180 (ITS), MW800361 (LSU), MW845677 (ACT) and MW892439 (TUB2). Blast search revealed that the ITS sequence had 100% (492/492 bp) homology with E. sorghinum LY-D-1-1, MT604999, LSU had 98% (1075/1091 bp) homology with E. sorghinum GZDS2018BXT010, MK516207, ACT had 96% (214/222 bp) homology with E. sorghinum M3, MK044832, and TUB2 had 99% (498/499 bp) homology with E. sorghinum BJ-F1, MF987525. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-1774.9882) is shown in Figure 2. Bipolaris, Curvularia and Fusarium spp. found causing leaf spot on maize were included in the phylogenetic tree (Liu et al. 2021; Reyes Gaige et al. 2020; Chang et al. 2016). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem and leaves of 2-week old maize plants. A solution containing 1 × 108 spores/mL (20 µL) was injected in the wound, whereas sterilized ddH2O was used in the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity for 3 days, observing fast-growing necrotic lesions in both stem and leaves. The pathogen was recovered from the infected plants and its identity was confirmed by morphological and sequence analyses. Microscope observations indicated the presence of chlamydospores, oval conidia (3 × 5 µm) and round pycnidia (60-100 µm diameter), and agree with those previously reported for the morphology of E. sorghinum (Bao et al. 2019). During last 2 years, E. sorghinum was reported to cause leaf spot on a number of relevant agricultural crops in China, including taro, Brassica parachinensis, tea, rice and wheat (Du et al. 2020; Li et al. 2020; Liu et al. 2020a, 2020b), confirming the expansion and host promiscuity of this pathogen. The pathogen was also reported to cause leaf spot on maize in Brazil in 2004 (Do Amaral et al. 2004); however, this is the first report of E. sorghinum causing leaf sheath and leaf spot on maize in China. Maize an important agricultural crop in China with more than 168 million tons produced in 2019. The observed yield loss and disease incidence of the isolated strain suggest that E. sorghinum may be a threat to maize production in China.

Occurrence of root rot caused by Pythium aphanidermatum on mung bean (Vigna radiata) in China.

In Aug 2019, approximately 10% of mung bean plants at the experimental farm of the Jiangsu Academy of Agricultural Science (32.03 N; 118.88 E) showed symptoms of stunting and wilting. Brown and water-soaked stem lesions were often observed at the base of the diseased plants. In severe cases, the plants collapsed and cumulous aerial mycelia were visible on the basal stem surface (Figure S1 A). To identify the causal agent, a total of 20 tissue fragments (5 mm long) were excised from roots and basal stems of five symptomatic plants. The fragments were surface sterilized in 2% sodium hypochlorite solution then plated on 2.5% potato dextrose agar (PDA) plates containing 10 μg/mL pimaricin, 100 μg/mL ampicillin, 10 μg/mL rifampicin, and 10 μg/mL pentachloronitrobenzene (PARP; Beckerman et al. 2017). After 3-4 days incubation at 25oC in dark, 14 colonies with white and cumulous mycelia grew from the tissue pieces (named as JS19-1 to JS19-14). JS19-1 and JS19-2 were purified by hyphal tipping, then grown on PDA medium for 7 days for morphological observation using a compound microscope (Figure S1 B, C). Width of coenocytic hyphae ranged from 3.7 to 8.9 (avg. 6.1, n=20) μm. Terminal oogonia were globose and with a diameter of 13.8 to 25.8 (avg. 22, n=20) μm. Antheridia were barrel-shaped, and mostly intercalary, sometimes terminal. Most of antheridia were diclinous, with 6.2 to 12.5 (avg. 9.3, n=20) μm in width and 7.6 to 15.3 (avg. 12.8, n=20) μm in length. Oogonia were fertilized with one or two (rare) antheridia. Oospores were aplerotic, 10.1 to 23.5 (avg. 20.4, n=20) μm in diameter. Sporangia had terminal inflated hyphal branches (Figure S1 D, E). The two isolates were preliminary identified as Pythium aphanidermatum. For molecular identification, the sequences of internal transcribed spacer (ITS) rDNA, cytochrome oxidase subunit I (CoxI) (Robideau et al. 2011), and β-tubulin (Kroon et al. 2004) of JS19-1 were detected, and deposited in GenBank (MT949538, MT949539 and MT949540). The ITS and CoxI sequences were identical with P. aphanidermatum CBS28779 ITS (759/759 bp, HQ643439.1) and PYT01 CoxI (640/640 bp, MH760243.1) respectively, the β-tubulin sequence showed 99% (830/840 bp) similarity of P. aphanidermatum P2 (AY564048.1). Thus, JS19-1 was confirmed as P. aphanidermatum. To fulfill Koch's postulates, the pathogenicity of JS19-1 was tested using the procedure of Kiyoshi et al. (2021) with some modifications. Barley grains infested with JS19-1 were as inoculum and thoroughly mixed with potting mixture at a rate of 10% in volume. Six mung bean seeds were sown per pot and then grown in a greenhouse. Potting mixture with no inoculum was used as control. Three pots of replicate plants used for inoculation and control. After 3 weeks, emergence in the inoculated pots was 33% and symptoms of stunting and root rot similar to those in field were observed, while control plants were asymptomatic (FigureS1 F, G). P. aphanidermatum was successfully reisolated from symptomatic plants of both methods. The pathogenicity tests were repeated twice. P. aphanidermatum causes seed rot, pre- and postemergence damping-off, or stem/root rot of a wide range of industrial crops and vegetables (Liu et al, 2018). To our knowledge, this is the first report of P. aphanidermatum causing disease on mung bean in China. Since Phytophthora vignae (Sun et al, 2020) and P. myriotylum (Yan et al, 2021) have been reported causing mung bean root rot, integrated disease management should be adopted to reduce damage.

Phytophthora heterospora sp. nov., a New Pseudoconidia-Producing Sister Species of P. palmivora.

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica. Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora, indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

Acute CoVID-19-associated bilateral neuropathy of the abducens nerve in a child

We have presented the first description of acute bilateral neuropathy of the abducens nerve associated with COVID-19 in a child.A boy aged 2 years 2 months acutely developed bilateral convergent strabismus, the next day fatigue, drowsiness, and a single vomiting not associated with food intake, were joined. A differential diagnosis was performed between brainstem encephalitis, other organic (non-inflammatory) lesions of the brain stem, and idiopathic bilateral neuropathy of the abducens nerves. According to laboratory data, mild lymphocytosis, mild eosinophilia were detected, according to polymerase chain reaction DNA of the herpes virus type 1/2, cytomegalovirus, herpes virus type 6, Epstein-Barr, tick-borne encephalitis, borrelia, anaplasma, RNA ehrlichia were not detected. IgM and IgG to tick-borne encephalitis were not detected. Neurophysiological and neuroimaging examinations did not reveal any pathology. On the 6th day of the disease, SARS-CoV-2 RNA was detected by polymerase chain reaction of the nasopharynx and oropharynx discharge. The final diagnosis was formulated. — bilateral neuropathy of the abducens nerves. Bilateral convergent paralytic strabismus. Concomitant diagnosis: new coronavirus infection, subclini-cal form (positive by polymerase chain reaction in the discharge from the throat and nose of the 2019-nCoV coronavirus (SARS-CoV-2) RNA). Decrease in the severity of strabismus was noted over time against the background of the therapy.Clinicians dealing with the COVID-19 should also pay attention to its ophthalmological manifestations and take into account that it can debut with them, as was in our case.

Epigenetic responses to Phytophthora citrophthora gummosis in citrus

Studies show that DNA methylation is associated with plant immunity but little is known as to how this epigenetic mechanism assists plants in adjusting their responses to biotic stress, especially when interacting with an hemibiotrophic pathogen such as citrus Phytophthora. The aim of the present study was to assess the effects of scion-rootstock interaction on plant resistance to P. citrophthora infection and DNA methylation patterns in ‘Pera’ sweet orange and ‘Tahiti’ acid lime grafted onto ‘Rangpur’ lime and ‘Tropical’ sunki rootstocks reinoculated with P. citrophthora. Results showed that reinoculated plants of the ‘Pera’ sweet orange/‘Rangpur’ lime and ‘Tahiti’ acid lime/‘Tropical’ sunki combinations with more and less sensitive varieties to Phytophthora, presented smaller stem lesions and increased frequency of full methylation and hemimethylation rates, compared to inoculated plants. In contrast, ‘Tahiti’ acid lime/‘Rangpur’ lime, two highly sensitive varieties, and ‘Pera’/‘Tropical’ sunki, two much less sensitive varieties, showed high increases in the frequency of hemimethylation and non-methylation levels. Results suggest that in citrus, both the scion-rootstock interaction and DNA methylation affect the response to P. citrophthora infection. Reinoculated plants, depending on the combination, showed changes in intracellular hyphae growth through the formation of sets of fibers and crystal accumulation in the periderm, cortex, and phloem. In addition, starch grain concentration was higher in reinoculated plants in comparison to inoculated plants. These findings support the assumption that DNA methylation is a plant defense mechanism and therefore may be exploited to improve the response of plants to the gummosis of P. citrophthora in citrus.

First report of soybean stem blight caused by Diaporthe phaseolorum in Sichuan province, China.

In September 2020, a disease resembling stem blight on soybean was found in Chengdu city, Sichuan province, southwestern of China. Symptoms began as a brown lesion on the stems, usually at the nodes, then lesions expanded, darkened, and even girdled the stems, causing wilt of the above stems (Figure 1A). In three 0.33-ha fields, a total of 300 soybean plants (20 plants/site × 5 sites/filed × 3 fields) were investigated, and 3% of the plants showed the disease symptoms. The symptoms were consistent with those previously reported for stem canker and stem blight on soybean caused by Diaporthe complex (Cui et al. 2009; Mena et al. 2019; Santos et al. 2011). The tissues of symptomatic soybean stems were rinsed by water, disinfected by submerging them in 75% ethanol for 30 s and in 2% sodium hypochlorite solution for 2 min, then followed by washing with sterile distilled water. Small diseased tissue fragments were placed on selective potato dextrose agar (PDA) containing rifampicin and ampicillin (both 50 mg/μl). Plates were sealed and incubated at 26°C for two days. Developing mycelia of isolates were transferred to fresh PDA and purified by single hyphal tip. For the five obtained isolates (Figure 1B), five markers, including the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, parts of the translation elongation factor 1-α (TEF1), part of the histone H3 (HIS) gene, the calmodulin gene (CAL), and the beta-tubulin gene (TUB), were amplified using the established primers ITS4/ITS5 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F (Crous et al. 2006) and H3-1b (Glass and Donaldson 1995), CAL228F/CAL737R (Carbone and Kohn 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995), respectively, and sequenced (GenBank IDs: MW595761-MW595780 and MW624472-MW624476). Phylogenetic trees were constructed based on concatenated sequences of the five markers using the maximum-likelihood (ML) method in MEGA-5.2.2. Based the results of morphological (Figure 1C-E) and phylogenetic analysis (Figure 2), the five isolates were all identified as D. phaseolorum. Pathogenicity tests for the isolates were conducted on 7-day-old soybean seedlings (cv. Shangdou 1310) using a hypocotyl slit inoculation method. At the stem 2-cm below cotyledon, a 6-mm long slit was cut with a sterile scalpel, and placed inside with a 3 mm × 3 mm PDA plug with or without mycelia of pathogen. Ten plants were assayed for each treatment, and the plants were maintained in greenhouse at 26°C, with humidity higher than 90% for the first two days. The assay was repeated at least three times. Typical brown lesions on the stems were observed four days after inoculation (Figure 1F), even 20% treated plants died. The D. phaseolorum was reisolated from these stem lesions. No disease symptom was observed on control plants (Figure 1G). Thus D. phaseolorum was the pathogen causing the soybean stem blight in field. To our knowledge, this is the first report of D. phaseolorum causing this disease in Sichuan province, China. The result may provide useful information for soybean disease control in this region of China. The authors declare no conflict of interest. Funding: China Agriculture Research System (CARS-004-PS14).

Effect of Different Botanical Extracts and Organic Compound in The Management of Leaf Spot Disease of Strawberry (Fragaria ananassa Duch.) Caused by Pestalotia longisetula under Field Condition

A field experiment was carried out using sweet sensation variety of strawberry to check the efficacy of different botanical extracts and organic compounds to manage leaf spot disease. The study was done using ten treatments viz; ginger (T1), turmeric (T2), garlic (T3), ginger + turmeric + garlic (T4), cow urine (T5), cow urine+ ginger + turmeric + garlic (T6), compost tea (T7), compost tea+ ginger+ turmeric+ garlic (T8), SAAF (mancozeb 63 % + carbendazim 12%) (T9) and control (T10), which was replicated thrice. The parameter observed during experiment were plant height, leaf number, disease leaves, stem lesions, yield, percent disease incidence, disease severity percent and percent disease control. At 75 days of transplantation, the highest plant height, leaf number (24.60) and yield (1391.67 gm/plot) were observed in treatment compost tea (18.14 cm), cow urine+ ginger+ turmeric+ garlic and garlic respectively and lowest in treatment control (16.19 cm, 23.27 &amp; 566.67 gm/plot). The disease leaves and stem lesions were observed highest in treatment control (3.40&amp;3.23) and lowest in treatments garlic (3.0), and cow urine+ ginger+ turmeric+ garlic (1.83) respectively at 75 days after transplantation. Moreover, the highest percent disease incidence and disease severity percent were observed highest in treatments control (28.94% &amp; 84.81%) and percent disease control in treatment garlic (42.36%) at 75 days of transplantation. Therefore, it is suggested to use garlic extracts as a measure to control leaf spot disease of strawberry. Int. J. Appl. Sci. Biotechnol. Vol 9(3): 193-202.

Mangrove Dieback and Leaf Disease in Sonneratia apetala and Sonneratia caseolaris in Vietnam

Even though survival rates for mangrove restoration in Vietnam have often been low, there is no information on fungal pathogens associated with mangrove decline in Vietnam. Therefore, this research was undertaken to assess the overall health of mangrove afforestation in Thanh Hoa Province and fungal pathogens associated with tree decline. From a survey of 4800 Sonneratia trees, the incidence of disorders was in the order of pink leaf spot &gt; shoot dieback &gt; black leaf spot for S. caseolaris and black leaf spot &gt; shoot dieback &gt; pink leaf spot for S. apetala. Approximately 12% of S. caseolaris trees had both pink leaf spot and shoot dieback, while only 2% of S. apetala trees had black leaf spot and shoot dieback. Stem and leaf samples were taken from symptomatic trees and fungi were cultured in vitro. From ITS4 and ITS5 analysis, four main fungal genera causing leaf spots and shoot dieback on the two Sonneratia species were identified. The most frequently isolated fungal taxa were Curvularia aff. tsudae (from black leaf spot),Neopestalotiopsis sp.1 (from stem dieback), Pestalotiopsis sp.1 (from pink leaf spot), and Pestalotiopsis sp.4a (from black leaf spot). The pathogenicity of the four isolates was assessed by under-bark inoculation of S. apetala and S. caseolaris seedlings in a nursery in Thai Binh Province. All isolates caused stem lesions, and Neopestalotiopsis sp.1 was the most pathogenic. Thus, investigation of fungal pathogens and their impact on mangrove health should be extended to other afforestation projects in the region, and options for disease management need to be developed for mangrove nurseries.