SDH Genes Research Articles

Resistance risk assessment of Rhizoctonia solani to four fungicides.

Hexaconazole, thifluzamide, difenoconazole and azoxystrobin are widely used fungicides for the control of Rhizoctonia solani in China. However, few studies have assessed the sensitivity and resistance risk of R. solani to these four fungicides. The sensitivities of 126 R. solani isolates to hexaconazole, thifluzamide, difenoconazole and azoxystrobin were determined, with average half maximal effective concentration (EC50) values of 0.0386, 0.0659, 0.663 and 1.508 μg mL-1, respectively. Field resistance monitoring of the four fungicides showed that the three isolates had moderate resistance to difenoconazole. Resistant mutants to the four fungicides were obtained by fungicide adaptation, and resistance could be stably inherited by most mutants. Compared with those of the parent isolates, the biological characteristics of hexaconazole-resistant mutants exhibited enhanced or similar compound fitness index (CFI), whereas most of the other mutants displayed reduced or comparable CFI. There was evidence of positive cross-resistance between hexaconazole and difenoconazole. In the presence of fungicides, the expression of the CYP51 genes in hexaconazole- and difenoconazole-resistant mutants significantly increased, the expression of SDH genes in thifluzamide-resistant mutants significantly decreased, and the expression of the Cyt b gene in azoxystrobin-resistant mutants did not significantly change. Based on these data, we speculated that R. solani had a low-to-medium resistance risk to four fungicides. The change of target gene expression may be one of the reasons for fungicide resistance in R. solani. This study provides a theoretical basis for monitoring resistance emergence and developing resistance management strategies to control R. solani. © 2024 Society of Chemical Industry.

Genetic Variation and Metabolic Basis of Kidney Cancer: New Opportunities for Targeted Therapy

Renal cell carcinoma (RCC) has previously been considered as a single disease. However, it is currently characterized as a heterogeneous group of tumors that differ in histological features, genetic abnormalities, and variable clinical course. In normal cells, energy is produced by the cleavage of chemical bonds in nutrients through the oxidation of fats, proteins, or carbohydrates. Mutational alterations in genes associated with RCC, including VHL, FLCN, PTEN and SDH, lead to abnormal cellular adaptation to changes in oxygen status, iron metabolism and nutrients. The present paper reviews the known genetic abnormalities observed in RCC and their impact on metabolic alterations. Understanding the genetic and metabolic mechanisms underlying RCC is crucial for the development of effective therapies. Targeting specific genetic abnormalities or metabolic pathways represents a promising approach to the RCC treatment. In addition, studies into the metabolic basis of RCC contribute to the development of new biomarkers for early diagnosis and monitoring of the disease. Moreover, investigating the role of VHL, FLCN, PTEN, and SDH genes in the development of RCC provides valuable information on the molecular mechanisms behind the disease. As a result, it may lead to the development of new treatment strategies aimed at restoring the normal function of these genes or compensating for their abnormalities. Overall, an integrated approach to the study of RCC that considers genetic, metabolic, and clinical aspects will ensure that more effective treatments are developed and prognosis for patients with this disease are improved.

Keratin 20 positive SDH-deficient renal cell carcinoma: a case report and literature review

This study aims to broaden the morphological scope of SDH-deficient renal cell carcinoma and to assist clinicians and pathologists in better understanding this entity to prevent misdiagnosis. This study used immunohistochemistry staining and the first-generation sequencing Sanger method for gene detection. It retrospectively analysed the clinical pathology, molecular characteristics, biological behaviour, and treatment information of one case of SDH-deficient renal cell carcinoma. The patient was a 57-year-old female with right back pain for more than 20 days and had no personal or family history of kidney tumours. In addition, the tumour cells had clear boundaries in morphology, and residual normal renal tubules could be seen around them. There were also ossification and adipose tissue around the tumour centre. The tumour cells were arranged in a glandular tubular and cord-like manner. Vacuolar and eosinophilic inclusion bodies could be observed in the cytoplasm. The nucleus was regular, the chromatin distribution was fine, and there were no obvious nucleoli. They were low-grade nuclei. In addition, no atypical mitosis or necrosis could been found. Furthermore, immunohistochemistry staining showed SDHB-negative and keratin 20 -positive tumour. Meanwhile, the first-generation sequencing also pointed out the presence of SDHB gene mutations in the tumour. After 12 months of follow-up, there was no evidence of disease recurrence in the patient. SDH-deficient renal cell carcinoma is a rare tumour associated with SDH gene germline mutations, and suspected cases should undergo SDHB immunohistochemistry staining. Most SDH-deficient renal cell carcinomas have a good prognosis, but undifferentiated cases require long-term follow-up.

8275 Recurrent Retroperitoneal Paraganglioma

Abstract Disclosure: J. Kang: None. V. Gupta: None. J.S. Subauste: None. Paragangliomas are rare, highly vascular neuroendocrine neoplasms that may originate from sympathetic or parasympathetic paraganglia and may secrete catecholamines. They are closely related to pheochromocytomas which are considered to be their intra-adrenal counterparts. A 65-year-old African American male with type 2 diabetes and tobacco use presented to his primary care physician with night sweats and unintentional weight loss of about 30 pounds in 3 months. Review of systems and laboratory workup was unremarkable. CT Chest/Abdomen/Pelvis with contrast showed a homogenous, well-circumscribed 4.6cm retroperitoneal mass causing mass effect on portal vein, Inferior vena cava, and right renal artery. Interventional radiology performed fine needle aspiration with intra-procedural blood pressure rising to 201/70 mmHg. Pathology showed paraganglioma, staining positive for synaptophysin, chromogranin A, CD 56 and S-100 protein. Plasma metanephrines were elevated at 2818 pg/mL, plasma normetanephrines at 1247 pg/mL with consistent 24-hour urine results. Workup for other adrenal hormones was negative. No family history of pheochromocytoma/paraganglioma or any clinical manifestations of Von-Hippel-Lindau syndrome or Neurofibromatosis were present. He was started on alpha blockade followed by beta blocker and surgical resection was planned. In the interim, escalating requirements of insulin were noted. Intra-operative course was complicated by systolic blood pressure rising to 290s which resolved after mass was removed. Immunohistochemical studies sent to Mayo, Rochester showed retained cytoplasmic SDHB expression in tumor cells. Hence, no further genetic testing was performed. Post excision, plasma-free metanephrines normalized with significant improvement in blood pressure and diabetes. He was followed with yearly plasma-free metanephrines for surveillance. Although asymptomatic, a three-fold rise of plasma metanephrines was noted five years later. CT Abdomen was concerning for recurrence. Dotate/PET scan showed dotate-avid para-aortic soft tissue lesion measuring 2.3 cm between the celiac artery and the superior mesenteric artery which was surgically excised with pathology consistent with paraganglioma (Ki67<1%). Diagnosis of paragangliomas is often in 3rd - 5th decade. About 30% are hereditary and associated with Von-Hippel-Lindau, Neurofibromatosis-1, MEN2A/B among other syndromes. They are mostly benign but SDHB pathogenic variants are likely to be malignant. Genetic testing should be considered in all paraganglioma patients to screen for germline pathogenic variants in SDH and other genes. Diagnosis of secretory paraganglioma is by urinary and/or plasma fractionated metanephrines and catecholamines. Treatment of choice is surgery when amenable to resection with preoperative medical blockade to prevent perioperative complications. Presentation: 6/1/2024

7553 Thyroid Paraganglioma: A Complex Tale of the Familiar Thyroid Nodule

Abstract Disclosure: V. Moorthy: None. A. Kondapally: None. L.E. Kimball-Ravari: None. Background: Thyroid paragangliomas are rare and often misdiagnosed on fine-needle aspiration (FNA). Clinical Case & Discussion: A 58-year-old female presented with an asymptomatic “cold” thyroid nodule. Ultrasound demonstrated a 2 cm hypoechoic nodule in the isthmus. FNA yielded a follicular lesion of undetermined significance, and genomic sequencing estimated a 50% risk of malignancy prompting the patient to opt for total thyroidectomy. Histopathology and immunohistochemistry (IHC) confirmed the diagnosis of benign paraganglioma (PGL). SDH-B (succinate dehydrogenase subunit B) was notably absent on IHC. Past medical history is significant for hysterectomy for uterine fibroids and obesity, along with a confirmed family history of Hereditary Leiomyomatosis with Renal Cell Cancer (HLRCC). The patient and several family members tested positive for a pathogenic variant of fumarate hydratase (FH) gene implicated in HLRCC and undergo annual MRIs for cancer surveillance. The loss of SDH-B suggests underlying mutation. Germline SDH mutations occur in 20% of patients with pheochromocytoma and PGLs, predisposing to hereditary syndromes. PGLs are catecholamine-secreting neuroendocrine tumors with carotid body and jugular PGLs accounting for 80% of head and neck PGLs (HNPGLs). Thyroid PGLs originate from the inferior laryngeal paraganglia. They are very rare, often non-secretory, and commonly present as asymptomatic solitary nodules. For these reasons, FNA, while contraindicated, is frequently performed. If FNA is considered, pre-procedural alpha-adrenergic blockade is recommended, although catecholamine secretion triggered by FNA is uncommon. Thyroid PGLs are misdiagnosed on FNA as other thyroid neoplasms. Awareness of the more unique cytopathology of thyroid PGLs and a comprehensive IHC can aid in accurate diagnosis. Interestingly, SDH (complex 2) and FH are closely linked enzymes in the Krebs cycle. Emerging evidence correlates germline FH mutations and hereditary pheochromocytoma-paraganglioma. PGLs are less commonly associated with MEN 2A and 2B, NF type 1, VHL, and Carney-Stratakis syndrome and Carney triad. Therefore, additional testing for SDH, RET, VHL and MAX genes was recommended in this case. Total thyroidectomy or lobectomy with or without elective lymph node dissection remains the treatment of choice. Annual 24-hour urine and plasma metanephrine testing will help screen for potential recurrence. Use of GLP1 agonists for weight loss in this patient warrants further genetic and safety evaluation. Clinical Lesson/Conclusion: Often misdiagnosed on FNA, thyroid paragangliomas are rare, with multiple genetic predispositions. FNA is contraindicated and if considered, must be performed after alpha-blockade. Germline SDH mutations are implicated in PGLs and increasing evidence links FH mutations to hereditary PGLs. Presentation: 6/3/2024

The New Nematicide Cyclobutrifluram Targets the Mitochondrial Succinate Dehydrogenase Complex in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus. The result shows that cyclobutrifluram is the most effective agent, with an LC50 value of 0.1078 mg·L-1. At an LC20 dose, it significantly reduced the population size to 10.40 × 103 ± 737.56-approximately 1/23 that of the control group. This notable impact may stem from the agent's ability to diminish egg-laying and hatching rates, as well as to impede the nematodes' development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc-dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

Association of a high rate of glycolysis and the activity of the uncoupling of oxidation and phosphorylation in the blood cells of patients with late-stage knee osteoarthritis and the development of postoperative pain

Objective: to investigate the relationship between the expression of genes mediating cellular energy production and the development of chronic postoperative pain (CPP) after total knee arthroplasty (TKA) in patients with osteoarthritis (OA).Material and methods. Prior to TKA, the blood of 50 patients with stage III–IV knee OA and complaints of constant pain and joint dysfunction was analyzed. The control group consisted of 26 healthy individuals. Pain intensity was assessed using a visual analogue scale (VAS), a short BPI questionnaire and the WOMAC index, and the presence of neuropathic pain was assessed using the DN4 and PainDETECT questionnaires. The development of CPP was determined 3 and 6 months after TKA. Total RNA isolated from blood was used to determine the expression of PKM2, LDH, SDH, AMPKα, PDH, IDH, MDH and ATP synthase genes by real-time quantitative reverse transcriptase-polymerase chain reaction.Results and discussion. CPP ≥30 mm according to VAS was detected in 17 patients. Before TKA, the expression of all analyzed genes was significantly increased compared to that of the control group. However, there were no differences in clinical, pain-related and functional indicators in the analyzed group of patients with OA. Before surgery, patients who subsequently developed CPP had significantly higher expression of genes related to glycolysis (PKM2, LDH), Krebs cycle – KC (SDH) and master regulator of energy metabolism (AMPKα) than patients who were satisfied with the results of TKA. At the same time, no differences were found in the expression of PDH and other KC enzyme genes (IDH, MDH) and ATP synthase in patients with and without CPP.Conclusion. The development of CPP is associated with a higher rate of glycolysis and energy deficiency, presumably due to the higher uncoupling activity of oxidation and phosphorylation that can be observed before TKA.

Efficacy of cyclobutrifluram in controlling Fusarium crown rot of wheat and resistance risk of three Fusarium species to cyclobutrifluram

Cyclobutrifluram (TYMIRIUM® technology), a new succinate dehydrogenase inhibitor (SDHI) fungicide, is currently being registered by SYNGENTA for controlling Fusarium crown rot (FCR) of wheat in China. The application of 15 or 30 g of active ingredient/100 kg seed of cyclobutrifluram significantly reduced pre-emergence damping-off, discoloration on the stem base and formation of whiteheads caused by FCR. The EC50 values of cyclobutrifluram for 60 isolates of F. pseudograminearum, 30 isolates of F. asiaticum and 30 isolates of F. graminearum ranged from 0.016 to 0.142 mg L−1, 0.010 to 0.041 mg L−1 and 0.012 to 0.059 mg L−1, respectively. One hundred and seven cyclobutrifluram-resistant (CR) mutants were obtained from three Fusarium species isolates, with ten types of mutations identified in Sdh genes. Three Fusarium species isolates exhibited similar resistance mechanisms, with the most prevalent mutations, SdhC1A83V and SdhC1R86K, accounting for 61.68% of mutants. The CR mutants possessed comparable or slightly impaired fitness compared to the corresponding parental isolates. The CR mutants carrying FpSdhBH248Y/Q/D exhibited increased sensitivity to fluopyram. An overall moderate risk of resistance development in three Fusarium species was recommended for cyclobutrifluram.

Sensitivity of tomato leaf mould–causing Fulvia fulva to seven succinate dehydrogenase inhibitor (SDHI) fungicides in Nara Prefecture, Japan and high efficacy of isofetamid in controlling SDHI‐resistant isolates

AbstractLeaf mould caused by Fulvia fulva is a major disease of tomato in Japan. Resistance to several types of fungicides, including succinate dehydrogenase inhibitors (SDHIs), has been reported in isolates from Japan. In this study, we evaluated the sensitivity of F. fulva to SDHI fungicides, SDHI fungicide efficacy in controlling sensitive and resistant isolates, and the presence of mutations in the Sdh B gene, the target site of SDHI fungicides. The minimum inhibitory concentration (MIC) of six SDHI fungicides, excluding isofetamid, ranged from 0.1 to >100 μg/mL against mycelial growth of 45 F. fulva isolates collected in Nara prefecture, Japan, whereas that of isofetamid ranged from 0.1 to only 10 μg/mL. The peak MIC distribution of fluopyram and isofetamid was 10 μg/mL, that of inpyrfluxam was 100 μg/mL and was >100 μg/mL for the remaining SDHI fungicides. In a greenhouse inoculation experiment, only isofetamid controlled the highly resistant isolates with an MIC of 10 μg/mL and the remaining fungicides had MIC >100 μg/mL. In addition, moderately and highly resistant isolates were controlled by the application of isofetamid 6 h or 7 days before inoculation or 3 days after inoculation. Mutations in the Sdh B gene were not observed among sensitive, moderately resistant and highly resistant isolates, suggesting that mutations in other Sdh genes and/or changes in efflux pump expression were involved. Our results indicate the presence of cross‐resistance and different levels of sensitivity reduction among SDHI fungicides. Isofetamid showed the lowest resistance development among the tested SDHI fungicides and is considered effective in controlling tomato leaf mould.

Physiological and biochemical characteristics of boscalid resistant isolates of Sclerotinia sclerotiorum from asparagus lettuce

Laboratory mutants of Sclerotinia sclerotiorum (Lib) de Bary, resistant to boscalid, have been extensively characterized. However, the resistance situation in the lettuce field remains largely elusive. In this study, among the 172 S. sclerotiorum isolates collected from asparagus lettuce field in Jiangsu Province, China, 132 isolates (76.74%) exhibited low-level resistance to boscalid (BosLR), with a discriminatory dose of 5 μg mL−1. In comparison to the boscalid-sensitive (BosS) isolates, most BosLR isolates demonstrated a slightly superior biological fitness, as evidenced by data on mycelial growth, sclerotium production and pathogenicity. Moreover, most BosLR isolates showed comparable levels of oxalic acid (OA) accumulation, increased exopolysaccharide (EPS) content and reduced membrane permeability when compared to the BosS isolates. Nevertheless, their responses to distinct stress factors diverged significantly. Furthermore, the effectiveness of boscalid in controlling BosLR isolates on radish was diminished compared to its efficacy on BosS isolates. Genetic mutations were identified in the SDH genes of BosLR isolates, revealing the existence of three resistant genotypes: I (A11V at SDHB, SDHBA11V), II (Q38R at SDHC, SDHCQ38R) and III (SDHBA11V+SDHCQ38R). Importantly, no cross-resistance was observed between boscalid and other fungicides such as thifluzamide, pydiflumetofen, fluazinam, or tebuconazole. Our molecular docking analysis indicated that the docking total score (DTS) of the type I resistant isolates (1.3993) was lower than that of the sensitive isolates (1.7499), implying a reduced affinity between SDHB and boscalid as a potential mechanism underlying the boscalid resistance in S. sclerotiorum. These findings contribute to an enhanced comprehension of boscalid’s mode of action and furnish valuable insights into the management of boscalid resistance.

Genotype-phenotype associations in paragangliomas of the temporal bone in a multi-ethnic cohort

Background Temporal bone paragangliomas are rare tumours with variable presentation that can be hereditary. Identification of clinical and genetic factors of aggressive tumour behaviour is important. Objective To determine the underlying genetic mutations and genotype/phenotype correlations in a multi-ethnic population of South Florida with sporadic temporal bone paragangliomas. Methods In a cohort of glomus tympanicum (GT) and glomus jugulare (GJ) cases, we assessed the frequency of pathogenic single nucleotide variants, insertions, deletions, and duplications in coding exons of genes that have been associated with paragangliomas (SDHB, SDHC, SDHD, SDHA, SDHAF2, RET, NF1, VHL, TMEM127, and MAX). Results None of the 12 GT cases had mutations. Among 13 GJ cases, we identified four mutation carriers (31%); two in SDHC, one in SDHB, and one in SDHD. All patients with pathogenic mutations were of Hispanic ethnicity, presented at a younger age (mean 27.5 versus 52.11 years), and with more advanced disease when compared to mutation-negative GJ cases. Conclusions and Significance: Mutations in the SDH genes are found in 31% of sporadic GJ. SDH-associated GJ had advanced disease and a 50% risk of metastasis. Our data supports emerging recommendations for genetic screening in all populations with GJ tumours as the genetic status informs management.

Longitudinal follow-up and outcomes of pediatric and adult patients with SDH-deficient GIST.

11542 Background: Gastrointestinal stromal tumor (GIST) are gastrointestinal non-epithelial neoplasms most commonly driven by somatic mutations in KIT or PDGFRA. Approximately ten percent of GIST are due to germline mutations in SDHx or epigenetic loss of expression of SDHC. While KIT and PDGFRa driven GIST respond to treatment with tyrosine kinase inhibitors, effective systemic therapies for SDH-deficient GIST have not yet been identified. SDH-deficient GIST are indolent tumors that typically progress slowly over time. A better understanding of the natural history of patients with these neoplasms is critical to identify effective treatments and improve patient care. A cohort of patients with SDH-deficient GIST is followed at the NIH Clinical Center and through the NIH Pediatric and Wild-type GIST Clinic. The aim of this study is to characterize the long-term outcome of this cohort of patients. Methods: Data from patients with SDH-deficient GIST enrolled in a study Natural History and Biospecimen Acquisition for Children and Adults with Rare Solid Tumors (NCT03739827) from January 2019 through January 2023 at the NIH were evaluated. In addition to review of medical records and imaging, when available, tumors were characterized by sequencing of SDH genes. Germline analysis of SDH genes was offered to consenting patients and families. Results: Clinical information and specimens were collected from 77 GIST patients (median age at diagnosis 21.5, [range 7-57] years; 72.7% (56) female, 21.3% (21) male) were classified by molecular subtypes: 33.8% SDHC epimutation, 28.6% SDHA, 22.1% SDHB, 14.3% SDHC and 1.3% SDHD. Median age at presentation of SDHC epimutation was younger (14.5 years, range: 8-56) when compared to SDHA (27.5 years, range: 7-55), SDHB (22 years, range: 8-54), SDHC (18 years, range: 10-57) and SDHD (39 years, N = 1). Most commonly, patients had at least one surgery (55.8%) while a lower proportion had four or more (7.8%). Primary tumors occurred in the stomach with 26% having metastases at presentation the most common location being the liver (10.4%). Overall, 64.9% of patients were treated with imatinib, 39% were treated with sunitinib and 15.6% received other additional systemic therapies. Within the cohort there were 13% of patients with paragangliomas and 6.5% with chondromas while only 9.1% had a family history of GIST. Conclusions: Longitudinal follow-up of an SDH-deficient GIST patient cohort will allow for better understanding of the natural history including treatment history and survival of patients with this rare disease and will help to optimize strategies for treatment and follow-up of these patients.

SDHI resistance in Pyrenophora teres f teres and molecular detection of novel double mutations in sdh genes conferring high resistance.

Net blotch (NB), caused by Pyrenophora teres f. teres (Ptt), is an important disease of barley worldwide. NB control is commonly achieved through the use of fungicide mixtures including strobilurins, triazoles and carboxamides. Succinate dehydrogenase inhibitors (SDHI) are important components of fungicide management programs of barley diseases. However, during the last growing seasons in Argentina, barley fields sprayed with mixtures containing SDHI fungicides have shown failures in NB control. Here, we report the isolation and characterization of Argentine Ptt strains resistant to SDHI fungicides. Compared against a sensitive (wild-type) reference strain collected in 2008, all 21 Ptt isolates collected in 2021 exhibited resistance to pydiflumetofen and fluxapyroxad both in vitro and in vivo. Concordantly, all of them presented target-site mutations in any of the sdhB, sdhC and sdhD genes. Although the mutations detected have been previously reported in other parts of the world, this study documents for the first time the occurrence of double mutations in the same Ptt isolate. Specifically, the double mutation sdhC-N75S + sdhD-D145G confers high resistance to SDHI fungicides, while the double mutations sdhB-H277Y + sdhC-N75S and sdhB-H277Y + sdhC-H134R confer moderate levels of resistance in Ptt. SDHI-resistance in Argentine Ptt populations is expected to increase. These findings emphasize the urgent need to perform a wider survey and a more frequent monitoring of SDHI sensitivity of Ptt populations and to develop and implement effective antiresistance tactics. © 2023 Society of Chemical Industry.

Experience with variants of the SDH gene related to paraganglioma in the Southern Health Area of Seville (Spain)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Metagenomics reveals the microbial community and functional metabolism variation in the partial nitritation-anammox process: From collapse to recovery

Mainstream partial nitritation-anammox (PNA) process easily suffers from performance instability and even reactor collapse in application. Thus, it is of great significance to unveil the characteristic of performance recovery, understand the intrinsic mechanism and then propose operational strategy. In this study, we combined long-term reactor operation, batch tests, and metagenomics to reveal the succession of microbial community and functional metabolism variation from system collapse to recovery. Proper aeration control (0.10-0.25 mg O2/L) was critical for performance recovery. It was also found that Candidatus Brocadia became the dominant flora and its abundance increased from 3.5% to 11.0%. Significant enhancements in carbon metabolism and phospholipid biosynthesis were observed during system recovery, and the genes abundance related to signal transduction was dramatically increased. The up-regulation of sdh and suc genes showed the processes of succinate dehydrogenation and succinyl-CoA synthesis might stimulate the production of amino acids and the synthesis of proteins, thereby possibly improving the activity and abundance of AnAOB, which was conducive to the performance recovery. Moreover, the increase in abundance of hzs and hdh genes suggested the enhancement of the anammox process. Changes in the abundance of key genes involved in nitrogen metabolism indicated that nitrogen removal pathway was more diverse after system recovery. The achievement of performance recovery was driven by anammox, nitrification and denitrification coupled with dissimilatory nitrate reduction to ammonium. These results provide deeper insights into the recovery mechanism of PNA system and also provide a potential regulation strategy for the stable operation of the mainstream PNA process.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

Combined evolutionary and metabolic engineering improve 2-keto-L-gulonic acid production in Gluconobacter oxydans WSH-004

The direct fermentation of the precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG), has been a long-pursued goal. Previously, a strain of Gluconobacter oxydans WSH-004 was isolated that produced 2.5 g/L 2-KLG, and through adaptive evolution engineering, the strain G. oxydans MMC3 could tolerate 300 g/L D-sorbitol. This study verified that the sndh–sdh gene cluster encoded two key dehydrogenases for the 2-KLG biosynthesis pathway in this strain. Then G. oxydans MMC3 further evolved through adaptive evolution to G. oxydans 2-KLG5, which can tolerate high concentrations of D-sorbitol and 2-KLG. Finally, by increasing the gene expression levels of the sndh–sdh and terminal oxidase cyoBACD in G. oxydans 2-KLG5, the 2-KLG accumulation in the 5-L fermenter increased to 45.14 g/L by batch fermentation. The results showed that combined evolutionary and metabolic engineering efficiently improved the direct production of 2-KLG from D-sorbitol in G. oxydans.

Resistance to pydiflumetofen in Botrytis cinerea: risk assessment and detection of point mutations in sdh genes that confer resistance.

Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study. A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 μg mL-1 . Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross-resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen-resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively. The fitness was significantly higher for all pydiflumetofen-resistant mutants than the corresponding parental. Two types of point mutations, sdhB-P225L and sdhC-G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry.

Transcriptome data-based identification of candidate genes involved in metabolism and accumulation of soluble sugars during fruit development in 'Huangguan' plum.

Fruit sweetness being an important factor of organoleptic quality directly affects the consumers' preferences for fresh fruit consumption, and is influenced by the composition and quantity of sugars. In this study, four soluble sugars (sucrose, fructose, glucose, and sorbitol) were identified and quantified in plum fruits cv. 'Huangguan' at four different maturity stages (fruitlet, green, veraison, and mature stage). The results revealed that sucrose and glucose are major soluble sugar components at the fruitlet and mature stages, respectively. RNA-Seq analysis was carried out and 6,778 differentially expressed genes (DEGs) were identified, including 121 genes involved in sugar metabolism. Furthermore, a total of 39 transcripts of 8 gene families encoding key enzymes related to the metabolism and accumulation of soluble sugars were separately identified. ERD6L (gene 103322904) was involved in keeping a balance of glucose between the inside and outside of vacuole. SS (gene 103333990) and SDH (gene 103335104) regulated the accumulation of fructose at the green stage. SDH (gene 103335104) controlled the degradation of sorbitol at the green stage. SS (gene 103333990) and PFK (gene 103333391) regulated the degradation of sucrose at the early stages of fruit development. Moreover, NINV (gene 103331108) regulated the accumulation of total sugar in plum. Genes 103321334 and 103335689 were important bZIP transcription factors that regulate the accumulation of glucose and fructose in fruits. Twelve DEGs were selected and validated to observe the relative accuracy of transcriptome sequencing data using qRT-PCR. Gene expression patterns were consistent between qRT-PCR and RNA-Seq data, indicating the reliability of RNA-Seq data. PRACTICAL APPLICATIONS: The results of this study provided new insights into comprehensive understanding of the genetic control of sugar metabolism and accumulation in plum fruits.

Genomics and proteomics combined analysis revealed the toxicity response of silkworm Bombyx mori to the environmental pathogen Bacillus cereus ZJ-4

Bacterial contamination has caused a major public health problem worldwide. Bacillus cereus is a conditional environmental pathogenic bacteria that can cause food poisoning. Whether environmental pathogens can cause widespread transmission in the insect kingdom is unclear. In this study, a Bacillus cereus ZJ-4 was isolated from the hospital environment of Zhenjiang City, Jiangsu Province, China. It was fatal by injection into the silkworm hemolymph. To investigated the potential toxic factors of ZJ-4 and clarified the toxicity response mechanism of silkworm by the ZJ-4 infection. Then, the whole genome of ZJ-4 was sequenced, and the immune mechanism of silkworm fat body to ZJ-4 pathogen was studied by HE pathological section and proteomics. Bacterial genome sequencing indicated that ZJ-4 had 352 drug resistance genes and 6 virulence genes. After 36 h of subcutaneous puncture with ZJ-4 suspension, the pathological changes were obviously found in HE pathological sections of fat body tissue. Comparative proteomic results indicated that differentially expressed proteins are mainly involved in stress reactions, biological regulation, and innate immunity. The qRT-PCR analysis showed that the expressions of β-GRP, Spaetzle, MyD88, Tube and Dorsal genes in Toll pathway were up-regulated, while Pell and Cactus genes were down-regulated; in the antimicrobial peptide pathway, Glv2, Lzm, Mor, and Leb3 genes were up-regulated, while attacin1 and defensin genes were down-regulated; Sod gene was up-regulated, while Cat gene was down-regulated in the antioxidant pathway; Ldh, Sdh, and Mdh genes were down-regulated in glucose metabolism pathway. These results indicated that ZJ-4 can damage the innate immune pathway of silkworm, and also affect the normal immune function of fat body cells.