Size Of Necrotic Lesions Research Articles

Evaluation of Safety and Efficacy of Cell Therapy Based on Osteoblasts Derived from Umbilical Cord Mesenchymal Stem Cells for Osteonecrosis of the Femoral Head: Study Protocol for a Single-Center, Open-Label, Phase I Clinical Trial.

Although mesenchymal stem cells (MSCs) insertion has gained recent attention as a joint-preserving procedure, no study has conducted direct intralesional implantation of human umbilical cord-derived MSCs (hUCMSCs) in patients with ONFH. This is a protocol for a phase 1 clinical trial designed to assess the safety and exploratory efficacy of human umbilical cord-derived osteoblasts (hUC-Os), osteogenic differentiation-induced cells from hUCMSCs, in patients with early-stage ONFH. Nine patients with Association Research Circulation Osseous (ARCO) stage 1 or 2 will be assigned to a low-dose (1 × 107 hUC-O cells, n = 3), medium-dose (2 × 107 cells, n = 3), and high-dose group (4 × 107 cells, n = 3) in the order of their arrival at the facility, and, depending on the occurrence of dose-limiting toxicity, up to 18 patients can be enrolled by applying the 3 + 3 escalation method. We will perform hUC-O (CF-M801) transplantation combined with core decompression and follow-up for 12 weeks according to the study protocol. Safety will be determined through adverse event assessment, laboratory tests including a panel reactive antibody test, vital sign assessment, physical examination, and electrocardiogram. Efficacy will be explored through the change in pain visual analog scale, Harris hip score, Western Ontario and McMaster Universities Osteoarthritis Index, ARCO stage, and also size and location of necrotic lesion according to Japanese Investigation Committee classification before and after the procedure. Joint preservation is important, particularly in younger, active patients with ONFH. Confirmation of the safety and efficacy of hUC-Os will lead to a further strategy to preserve joints for those suffering from ONFH and improve our current knowledge of cell therapy.

Tissue-Engineered Bone Regeneration for Medium-to-Large Osteonecrosis of the Femoral Head in the Weight-Bearing Portion: An Observational Study.

Stem cell therapy for the treatment of osteonecrosis of the femoral head (ONFH) showed promising outcomes. However, ONFH with a large lesion in the weight-bearing portion is a poor prognostic factor and still challenging issue to be solved. We aimed to evaluate the effect of tissue-engineered bone regeneration for this challenging condition to preserve the femoral head. A total of 7 patients (9 hips) with ONFH who received osteoblasts expanded ex vivo from bone marrow-derived mesenchymal stem cells (BMdMSCs) and calcium metaphosphate (CMP) as scaffolds from March 2002 to March 2004 were retrospectively reviewed. The median age was 27.0 years (interquartile range [IQR], 23.0-34.0 years), and the median follow-up period was 20.0 years (IQR, 11.0-20.0 years). After culture and expansion of stem cells, we performed core decompression with BMdMSC implantation at a median number of 10.1 ×107 (IQR, 9.9-10.9 ×107). To evaluate radiographic outcomes, the Association Research Circulation Osseous (ARCO) classifications, the Japanese Investigation Committee (JIC) classification, and modified Kerboul combined necrotic angle (mKCNA) were evaluated preoperatively and during follow-up. Clinical outcomes were evaluated by a visual analog scale (VAS) and Harris Hip Score (HHS). The preoperative stage of ONFH was ARCO 2 in 5 hips and ARCO 3a in 4 hips. The ARCO staging was maintained in 3 hips of ARCO 2 and 4 hips of ARCO 3a. Two hips of ARCO 2 with radiographic progression underwent total hip arthroplasty. According to mKCNA, 2 hips showed medium lesions, and 7 hips showed large lesions. The size of necrotic lesion was decreased in 4 hips (2 were ARCO 2 and 2 were ARCO 3a). There were no significant changes in JIC classification in all hips (type C1: 3 hips and type C2: 6 hips) (p = 0.655). Clinically, there were no significant changes in the VAS and HHS between preoperative and last follow-up (p = 0.072 and p = 0.635, respectively). Tissue engineering technique using osteoblasts expanded ex vivo from BMdMSC and CMP showed promising outcomes for the treatment of pre-collapsed and early-collapsed stage ONFH with medium-to-large size, mainly located in weight-bearing areas.

Three-dimensional mapping of necrotic lesions for early-stage osteonecrosis of the femoral head

BackgroundThere is a scarcity of evidence regarding the potential relationship between the size and location of necrotic lesions, which must be understood to provide optimal joint-preserving treatment. The purpose of this study was to characterize the distribution patterns of necrotic lesions of varying sizes in early-stage osteonecrosis of femoral head (ONFH) with the use of three-dimensional mapping.MethodsWe retrospectively evaluated clinical CT images of the hips that were performed in the Third Hospital of Hebei Medical University from January 2018 to December 2022 and collected all CT images diagnosed with stage I and II ONFH. Three-dimensional structures that included both necrotic lesions and normal areas of the femoral heads were reconstructed and divided into eight regions to record their size and location. CT images for all lesions were superimposed onto a standard template, and three-dimensional mapping was created to determine the presence of concentrated areas of lesions.ResultsIn a cohort of 143 patients with stage I and II ONFH, a total of 150 hips were reviewed. For lesions with less than 15% of the femoral head volume, necrotic lesions predominantly involve regions I, III, and V, with region I showing concentration. For lesions with volumes ranging from 15 to 30%, necrotic lesions exhibited a wider distribution across regions I, II, III, IV, V, and VII, with significant concentrations in regions I, III, and V. For lesions exceeding 30% of the femoral head volume, the necrotic lesions were extensively distributed across nearly the entire femoral head, with a notable expansion of the concentrated necrotic areas.ConclusionsThe distribution of necrotic lesions varies with lesion size, with smaller lesions primarily concentrated in the anterior and medial regions of the femoral head, particularly in the anterosuperior region, while larger lesions expand to the lateral and inferior regions. These findings enhance existing classification systems and provide crucial insights for guiding hip-preserving surgical planning and approaches.

Analysis of the antagonistic effect of Stenotrophomonas geniculata WXY53 on Magnaporthe oryzae through bioassays and Whole-Genome sequencing

Magnaporthe oryzae causes rice blast, which poses a significant threat to agriculture. The use of environmentally friendly biological control agents is one of the methods used to reduce disease incidence. Our study identified the isolate WXY53 as Stenotrophomonas geniculata using 16S rDNA and phylogenetic tree analysis. WXY53 exhibited a 53.39 % inhibition rate on the growth diameter of M. oryzae hyphae in the dual culture assay. The WXY53 fermentation broth treatment inhibited 97.89 % of appressoria formation and delayed the colonization of plant cells by invasive hyphae. Applying WXY53 fermentation broth had a significant impact on preventing rice blast in the greenhouse experiment. It suppressed the size of necrotic lesions and reduced the number of lesions in rice and barley by 66.25 % and 72.0 %, respectively. The whole-genome sequencing of WXY53 revealed a genome size of 4,504,923 bp, with 66.51 % GC content and 4038 protein-coding genes. The study utilized multiple databases, including the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, and Carbohydrate-Active enZYmes to annotate gene functions. The analysis revealed genes related to environmental information processing, biological processes, cell wall/membrane/envelope biogenesis, glycoside hydrolases, and metabolism. Antibiotics & Secondary Metabolite Analysis Shell analysis predicted that WXY53 has the potential to produce aryl polymers, entolysin, and 2,3-dihydroxybenzoyl serine, in addition to the three gene clusters with unknown functions. Based on these findings, strain WXY53 could be a promising novel source of biological agents for managing rice blast. This study provides a theoretical basis for further investigation into microbial resources and metabolic gene clusters for agricultural biological control.

Preliminary screening of citrus hybrids for identifying tolerance to Phytophthora nicotianae

Phytophthora is one of the most devastating pathogens affecting citrus production. Breeding resistant genotypes is a durable strategy to manage this pathogen. In this study, in vitro screening of 31 citrus hybrids was done against Phytophthora nicotianae using detached leaf assay. The hybrids tested were developed in twoway crosses between Rangpur lime (RL, Phytophthora sensitive) and sour orange (SO, Phytophthora tolerant). The relative Phytophthora sensitivity was determined from the necrotic lesion area after three days of pathogen inoculation. The parents and hybrids could be differentiated into five resistance classes on the basis of necrotic lesion size and area. Among the 18 SO × RL (SR) hybrids, three hybrids viz. SR 3, SR 10 and SR 19 exhibited the least leaf necrotic length (2.8-3.7 mm), breadth (3.2-3.9 mm) and area (9.2-15.0 mm2), indicating a highly resistant reaction. From the 13 RL × SO (RS) hybrids, RS 6 and RS 22 showed highly resistant reaction by virtue of the least necrotic area (12.7-17.9 mm2). Twelve SR and four RS hybrids produced necrotic lesions of >216.8 mm2 area and were rated as highly susceptible in this assay. The parents- Rangpur lime and sour orange recorded highly susceptible and resistant reaction, respectively, which authenticated the results of the leaf assay. The five highly resistant hybrids identified in this study after ex vitro validation of Phytophthora tolerance should prove helpful in commercial citriculture.

The efficiency of Thymus vulgaris, Lavandula angustifolia, and Cupressus sempervirens essential oils in suppression of Hymenoscyphus fraxineus on Fraxinus angustifolia seedlings

AbstractDue to intense ash dieback, this research tested the bioactivity of Thymus vulgaris, Lavandula angustifolia, and Cupressus sempervirens essential oils (EO) against Hymenoscyphus fraxineus. In vitro testing showed that EOs had a pronounced fungitoxic effect on H. fraxineus. The most effective EO was extracted from T. vulgaris, while EOs obtained from L. angustifolia and C. sempervirens were less effective and showed the same effect. At a concentration of 1%, all tested EOs inhibited mycelial growth, while 0.01% and 0.1% concentrations of EO extracted from T. vulgaris also inhibited the growth of H. fraxineus. The in planta test of EO extracted from T. vulgaris conducted on three‐year‐old Fraxinus angustifolia seedlings showed a reduction in the size of necrotic lesions and a better crown condition compared to untreated seedlings. This research demonstrates, for the first time, the efficiency of EOs in suppressing ash dieback making a path for the development of new biological protection measures against H. fraxineus.

Identification and Characterization of Fungi Associated with Leaf Spot Disease of Rubber Trees (Hevea brasiliensis) in Pahang, Malaysia

Rubber trees are important sources of agricultural income as they are utilized for lumber and latex. Like other crops, they are susceptible to numerous fungal pathogens, especially on their leaves, the most important plant part. Therefore, this study was conducted to identify the fungi associated with leaf spot disease on the leaves of rubber trees. This study aimed to characterize fungi associated with diseased rubber leaves and determine the causative agent of leaf spot disease. A total of 20 fungal isolates were obtained and purified from rubber leaves collected during sampling in rubber plantations in Pahang, Malaysia. All the isolated fungi were identified as Colletotrichum siamense (2 isolates), Diaporthe sp. (5 isolates), Lasiodiplodia pseudotheobromae (1 isolate), L. theobromae (7 isolates) and Neoscytalidium sp. (5 isolates). Based on a pathogenicity test, it was found that nine isolates were pathogenic towards the leaves of rubber, which were L. theobromae, L. pseudotheobromae, and C. siamense. The lesions on the leaves displayed chlorosis with varying necrotic lesion sizes after 21 days post-inoculation. The isolates of the species L. theobromae were the most prevalent, indicating it is more widespread in the region than other species. The findings present data on the potential pathogen of rubber plants and are important to understand further the potential risks and effects of the pathogen in rubber plantations

Isolation and Evaluation of Streptomyces melanogenes YBS22 with Potential Application for Biocontrol of Rice Blast Disease.

Plant diseases caused by pathogenic fungi pose a significant threat to agricultural production. This study reports on a strain YBS22 with broad-spectrum antifungal activity that was isolated and identified, and its active metabolites were purified and systematically studied. Based on a whole genome sequence analysis, the new strain YBS22 was identified as Streptomyces melanogenes. Furthermore, eight gene clusters were predicted in YBS22 that are responsible for the synthesis of bioactive secondary metabolites. These clusters have homologous sequences in the MIBiG database with a similarity of 100%. The antifungal effects of YBS22 and its crude extract were evaluated in vivo and vitro. Our findings revealed that treatment with the strain YBS22 and its crude extract significantly reduced the size of necrotic lesions caused by Magnaporthe oryzae on rice leaves. Further analysis led to the isolation and purification of an active compound from the crude extract of the strain YBS22, identified as N-formylantimycin acid methyl ester, an analog of antimycin, characterized by NMR and MS analyses. Consistently, the active compound can significantly inhibit the germination and development of M. oryzae spores in a manner that is both dose- and time-dependent. As a result, we propose that the strain YBS22 could serve as a novel source for the development of biological agents aimed at controlling rice blast disease.

Experimental Assessment of the Impact of Sonication Parameters on Necrotic Lesions Induced in Tissues by HIFU Ablative Device for Preclinical Studies

We have designed and built ultrasound imaging-guided HIFU ablative device for preclinical studies on small animals. Before this device is used to treat animals, ex vivo tissue studies were necessary to determine the location and extent of necrotic lesions created inside tissue samples by HIFU beams depending on their acoustic properties. This will allow to plan the beam movement trajectory and the distance and time intervals between exposures leading to necrosis covering the entire treated volume without damaging the surrounding tissues. This is crucial for therapy safety. The objective of this study was to assess the impact of sonication parameters on the size of necrotic lesions formed by HIFU beams generated by 64-mm bowl-shaped transducer used, operating at 1.08 MHz or 3.21 MHz. Multiple necrotic lesions were created in pork loin samples at 12.6-mm depth below tissue surface during 3-s exposure to HIFU beams with fixed duty-cycle and varied pulse-duration or fixed pulse-duration and varied duty-cycle, propagated in two-layer media: water-tissue. After exposures, the necrotic lesions were visualized using magnetic resonance imaging and optical imaging (photos) after sectioning the samples. Quantitative analysis of the obtained results allowed to select the optimal sonication and beam movement parameters to support planning of effective therapy.

Posterior Pelvic Tilt in the Standing Position Might Be Associated with Collapse Progression in Post-Collapse Stage Osteonecrosis of the Femoral Head.

Excessive pelvic tilt has been reported to impair the biomechanical loading of the hip joint. However, the influence of pelvic tilt in osteonecrosis of the femoral head (ONFH) remains unclear. This study aims to assess whether sagittal pelvic posture in the standing position correlates with progression of femoral head collapse in post-collapse stage ONFH. This is a single-center retrospective study. We investigated 107 patients (107 hips; 73 males and 34 females; mean age, 48 years) diagnosed with Association of Research Circulation Osseous (ARCO) stage III ONFH at the first visit and who subsequently underwent surgical treatment in our institution from July 2016 to December 2020. The sagittal pelvic posture in the standing position before surgery was quantified as the angle formed by the anterior pelvic plane and the vertical z-axis in the sagittal view (APP angle). An APP angle <0° indicated posterior pelvic tilt. Progression of femoral head collapse was calculated as collapse speed. The following factors potentially associated with collapse speed were evaluated by exploratory data analysis followed with multiple linear regression analysis: sex, age, BMI, etiology, pelvic incidence, contralateral hip condition, time interval between the first visit and surgery, size of necrotic lesion, location of necrotic lesion, and APP angle. As ONFH progressed from ARCO stage IIIA to stage IV, APP angle decreased significantly and continuously (stage IIIA, -0.2° ± 5.5°; stage IIIB, -3.7° ± 5.8°; stage IV, -7.1° ± 6.4°). The factors significantly associated with collapse speed were size of necrotic lesion (p=0.0079), location of necrotic lesion (p=0.0190), and APP angle (p < 0.0001). APP angle showed a negative correlation with collapse speed (r=-0.40, p < 0.0001). After stratifying by size of necrotic lesion (<50% and ≥50% involvement) and location of necrotic lesion (JIC type C1 and C2), a significant negative correlation was observed between APP angle and collapse speed in each group (JIC type C1 with <50% involvement, r=-0.69, p < 0.0001; JIC type C1 with ≥50% involvement, r=-0.58, p=0.0475; JIC type C2 with <50% involvement, r=-0.51, p=0.0124; JIC type C2 with ≥50% involvement, r=-0.39, p=0.0286). Our results suggest that posterior pelvic tilt in the standing position occurred as ONFH progressed from ARCO stage IIIA to stage IV, which might be associated with progression of femoral head collapse in ONFH.

Is There an Association Between Femoral Head Collapse and Acetabular Coverage in Patients With Osteonecrosis?

Osteonecrosis of the femoral head (ONFH) classification systems are based on the size, volume, and location of necrotic lesions. Often-but not always-ONFH results in femoral head collapse. Because acetabular coverage is associated with mechanical stress on the femoral head, it might also be associated with femoral head collapse in patients with ONFH. However, the association between acetabular coverage and femoral head collapse in these patients has not been established. (1) Is femoral head collapse associated with acetabular coverage or pelvic incidence (PI) in patients with ONFH? (2) Are established predictors of femoral head collapse in ONFH classification systems associated with acetabular coverage? Between 2008 and 2018, we evaluated 343 hips in 218 patients with ONFH. We considered all patients with ONFH except for those with a traumatic etiology, a history of surgical treatment before collapse, or those with collapse at initial presentation as potentially eligible for this study. Of those, 101 hips with ONFH (50% [50] were in males with a mean age of 44 ± 15 years) met our inclusion criteria. These patients were subsequently divided into two groups: those with femoral head collapse within 12 months (collapse group, 35 hips) and those without femoral head collapse (noncollapse group, 66 hips). No differences in patient demographics were observed between the two groups. CT images were used to measure the PI and acetabular coverage in three planes: the lateral center-edge angle (LCEA) in the coronal plane, the anterior and posterior center-edge angle in the sagittal plane, and the anterior and posterior acetabular sector angle in the axial plane; in addition, the difference between these parameters was investigated between the groups. The thresholds for femoral head collapse in the parameters that showed differences were investigated. Necrotic location and size were evaluated using the Japanese Investigation Committee (JIC) classification and the Steinberg grade classification, respectively. We examined the relationship between these parameters and classifications. The mean LCEA was slightly greater in the noncollapse group than in the collapse group (32° ± 6° versus 28° ± 7°; mean difference 4° [95% CI 1.15° to 6.46°]; p = 0.005); the clinical importance of this small difference is uncertain. There were no differences in PI between the two groups. After accounting for sex, age, BMI, and etiology as confounding factors, as well as acetabular coverage parameters and PI, we found a lower LCEA to be independently associated with increased odds of collapse, although the effect size is small and of questionable importance (OR 1.18 [95% CI 1.06 to 1.33]; p = 0.001). The threshold of LCEA for femoral head collapse was 28° (sensitivity = 0.79, specificity = 0.60, area under the curve = 0.73). The percentage of patients with an LCEA less than 28° was larger in JIC Type C1 (OR 6.52 [95% CI 1.64 to 43.83]; p = 0.006) and C2 (OR 9.84 [95% CI 2.34 to 68.38]; p = 0.001) than in patients with both Type A and Type B. The acetabular coverage data for the excluded patients did not differ from those of the patients included in the analysis. Our findings suggest that acetabular coverage appears to have little, if any, association with the likelihood of collapse in patients with ONFH. We found a small association between a lower LCEA and a higher odds of collapse, but the effect size may not be clinically important. Factors other than acetabular coverage need to be considered, and if our findings are verified by other investigators, osteotomy is unlikely to have a protective role. As the patients in our study were fairly homogeneous in terms of ethnicity and BMI, these factors need to be further investigated to determine whether they are associated with femoral head collapse in ONFH. Level III, prognostic study.

Multiomic approaches reveal novel lineage-specific effectors in the potato and tomato early blight pathogen Alternaria solani

The effectome of the necrotrophic fungal pathogen, Alternaria solani, was determined using multiomics. In total, 238 effector candidates were predicted from the A. solani genome, and apoplastic effectors constitute most of the total candidate effector proteins (AsCEPs). Comparative genomics revealed two main groups of AsCEPs: lineage-specific and conserved effectors. RNA-Seq analysis revealed that the most highly expressed genes encoding AsCEPs were enriched with lineage-specific forms. Two lineage-specific effector genes, AsCEP19 and AsCEP20, were found to form a ‘head-to-head’ gene pair located near an AT-rich region on the chromosome. To date, AsCEP19 and AsCEP20 have been found only in a few fungal species. Phylogenetic inference revealed that AsCEP19 and AsCEP20 were likely acquired by the common ancestor of A. solani and A. tomatophila via horizontal gene transfer, probably mediated by long terminal repeat retrotransposon. RT-qPCR analysis showed that AsCEP19 and AsCEP20 are tightly coexpressed in a host-specific manner and that they are upregulated at advanced stages of A. solani infection only in solanaceous hosts. Transient expression of AsCEP19 and AsCEP20 in Nicotiana benthamiana plants showed that these effectors could promote Phytophthora infestans infection. AsCEP19 and AsCEP20 were required for the full virulence of A. solani on host potato, because deletion of this gene pair significantly reduced the size of necrotic lesions on potato leaves. Transient expression of AsCEP20 could elicit plant cell death depending on the presence of its signal peptide, indicating that AsCEP20 is a necrosis-inducing apoplastic effector with the mature form localized specifically in chloroplasts. Our work provides a better understanding of the function and evolution of necrotrophic fungal effectors, and helps explain the high aggressiveness of A. solani against solanaceous crops.

Quantitative Magnetic Resonance Imaging of Femoral Head Articular Cartilage Change in Patients with Hip Osteonecrosis Treated with Extracorporeal Shock Wave Therapy.

Background Multiple reports have demonstrated the therapeutic potential of extracorporeal shock wave (ESWT) in osteonecrosis of the femoral head (ONFH). However, few studies reported the changes in hip articular cartilage after the intervention. This study aimed to investigate the effect of ESWT on femoral head cartilage using a novel technique, quantitative T2-mapping magnetic resonance imaging. Methods A total of 143 eligible patients with unilateral early-stage ONFH were randomized into the ESWT group and control group. Seventy-three patients in the ESWT group received two sessions of ESWT with oral drug treatment, while seventy patients in the control group received oral drug treatment only. The visual analog pain scale (VAS) and Harris hip score (HHS) at 3-month, 6-month, and 12-month follow-up were used as the clinical evaluation index. The radiological evaluation index used the T2 mapping values, necrotic size, and China-Japan Friendship Hospital (CJFH) classification. Results A total of 143 patients (62 females and 81 males) were finally included, and the characteristics before treatment were comparable between the two groups. At the last follow-up (12 months), the T2 values and ΔT2 changes in the ESWT group were all smaller than those in the control group (p=0.042; p=0.039), while the CJFH classification of ONFH and necrotic lesion size were not statistically significant. At 3 months and 6 months, the VAS in the ESWT group was lower than that in the control group (p=0.021; p=0.046) and the HHS in the ESWT group was higher (p=0.028; p=0.039). However, there were no significant differences in the VAS and HHS at 12 months between the ESWT and control groups. Conclusions The results of the current study indicated that, based on drug treatment, ESWT is an effective treatment method for nontraumatic ONFH, which could result in significant pain relief and function restoration. Furthermore, it could delay the injury of femoral head cartilage during the progression of ONFH.

Reliability and Repeatability of the China-Japan Friendship Hospital Typing Classification for Nontraumatic Osteonecrosis of the Femoral Head.

The purpose of this study was to analyze the interobserver reliability and intraobserver repeatability of the China-Japan Friendship Hospital (CJFH) typing classification system for nontraumatic osteonecrosis of the femoral head (ONFH). Image data of 50 hips were randomly selected in 289 patients (433 hips) with ONFH who underwent treatment in the China-Japan Friendship Hospital from 2012 to 2016. Bilateral hip plain radiography, magnetic resonance imaging (MRI), or computerized tomography (CT) were performed in all hips. The assessments were performed by 8 new residents independently, and the repeatability was assessed at 4-week intervals. Evaluation indicators included the size, location, and extent of necrotic lesions. Kappa values were used to determine the reliability and repeatability. According to the CJFH classification system, 2,800 evaluations were performed with an average interobserver Kappa value of 0.711, and 400 assessments were performed with an average intraobserver Kappa value of 0.748. Reliability analysis indicated a higher reliability and repeatability of this classification system. Critical factors affecting the consistency included the accurate selection of the median coronal plane and definitive tri-pillar division of the femoral head. The CJFH classification system is a simple and direct evaluation model for ONFH with substantial inter- and intraobserver reliability.

Postharvest Fumigation of (E)-2-Hexenal on Kiwifruit (Actinidia chinensis cv. ‘Haegeum’) Enhances Resistance to Botrytis cinerea

(E)-2-Hexenal is a reactive electrophile species released by green plants upon herbivory or pathogenic infection and induces various defense responses. In this study, (E)-2-hexenal was used as a natural alternative to artificial fumigants to prevent postharvest pathogenic infection of the ‘Haegeum’ kiwifruit (Actinidia chinensis). Integrated transcriptome-metabolome analyses were performed to investigate its effect on the induction of plant defense responses. When kiwifruit was fumigated with 100 μmol L-1 (E)-2-hexenal for 24 h at 20 °C and then inoculated with fungal pathogen Botrytis cinerea, the disease severity, as assessed by necrotic lesion size and B. cinerea biomass, was reduced compared to the control, distilled water-treated fruit. (E)-2-Hexenal treatment also led to upregulated expression of genes encoding pattern recognition receptors, signal transducer proteins, and pathogenesis-related proteins, including chitinase. Furthermore, treatment led to increased jasmonic acid and flavonoid biosynthesis. (E)-2-Hexenal fumigation therefore enhanced the resistance of kiwifruit to B. cinerea infection, presumably by facilitating the signaling network from pathogen recognition to defense gene expression and enabling the accumulation of secondary metabolites, such as flavonoids. The present study not only suggests an optimal concentration of (E)-2-hexenal fumigation for controlling postharvest disease of kiwifruit, but also broadens our understanding of the underlying molecular mechanism by which (E)-2-hexenal enhances plant resistance.

Gasdermin D mediates host cell death but not interleukin-1\u03b2 secretion in Mycobacterium tuberculosis-infected macrophages

Necrotic cell death represents a major pathogenic mechanism of Mycobacterium tuberculosis (Mtb) infection. It is increasingly evident that Mtb induces several types of regulated necrosis but how these are interconnected and linked to the release of pro-inflammatory cytokines remains unknown. Exploiting a clinical cohort of tuberculosis patients, we show here that the number and size of necrotic lesions correlates with IL-1β plasma levels as a strong indicator of inflammasome activation. Our mechanistic studies reveal that Mtb triggers mitochondrial permeability transition (mPT) and subsequently extensive macrophage necrosis, which requires activation of the NLRP3 inflammasome. NLRP3-driven mitochondrial damage is dependent on proteolytic activation of the pore-forming effector protein gasdermin D (GSDMD), which links two distinct cell death machineries. Intriguingly, GSDMD, but not the membranolytic mycobacterial ESX-1 secretion system, is dispensable for IL-1β secretion from Mtb-infected macrophages. Thus, our study dissects a novel mechanism of pathogen-induced regulated necrosis by identifying mitochondria as central regulatory hubs capable of delineating cytokine secretion and lytic cell death.

CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans.

Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.

The bZIP Transcription Factor LtAP1 Modulates Oxidative Stress Tolerance and Virulence in the Peach Gummosis Fungus Lasiodiplodia theobromae.

Lasiodiplodia theobromae is one of the primary causal agents in peach gummosis disease, leading to enormous losses in peach production. In our previous study, a redox-related gene, LtAP1, from the fungus was significantly upregulated in peach shoots throughout infection. Here, we characterized LtAP1, a basic leucine zipper transcription factor, during peach gummosis progression using the CRISPR-Cas9 system and homologous recombination. The results showed that LtAP1-deletion mutant had slower vegetative growth and increased sensitivity to several oxidative and nitrosative stress agents. LtAP1 was highly induced by exogenous oxidants treatment in the L. theobromae wild-type strain. In a pathogenicity test, the deletion mutant showed decreased virulence (reduced size of necrotic lesions, less gum release, and decreased pathogen biomass) on infected peach shoots compared to the wild-type strain. The mutant showed severely reduced transcription levels of genes related to glutaredoxin and thioredoxin in L. theobroame under oxidative stress or during infection, indicating an attenuated capacity for reactive oxygen species (ROS) detoxification. When shoots were treated with an NADPH oxidase inhibitor, the pathogenicity of the mutant was partially restored. Moreover, ROS production and plant defense response were strongly activated in peach shoots infected by the mutant. These results highlight the crucial role of LtAP1 in the oxidative stress response, and further that it acts as an important virulence factor through modulating the fungal ROS-detoxification system and the plant defense response.

A Novel Plant Resistance Inducer for the Protection of European Ash (Fraxinus excelsior L.) against Hymenoscyphus fraxineus—Preliminary Studies

Ash tree disease is caused by an ascomycete fungus Hymenoscyphus fraxineus, which first emerged in 1992, eastern Poland. Site factors, genetic predispositions, and resistance to the pathogen have not been fully described yet. The general aim of the study undertaken was to check the effect of using a new active substance representing benzothiadiazoles, a BTH derivative, namely, N-methyl-N-methoxyamide-7-carboxybenzo(1.2.3)thiadiazole (BTHWA), on ash saplings. A total of 41 ash saplings, aged three to five years, were subjected to this experiment in six variants of treatment. The results of the inoculation with H. fraxineus indicated that the treatment with BTHWA resulted in the limitation of the size of necrotic phloem lesions. Although the lesions were detectable in the cross section, the plants showed no visible signs of infection. The results suggest that H. fraxineus development in ash saplings can be slowed down or even completely stopped through triggering plant resistance by BTHWA.

Development of Hymenoscyphus fraxineus on seedlings from different half‐sib lines of Fraxinus angustifolia in Serbia

AbstractFor the long‐term goal of improving planting success and reforestation of floodplain forests, an underbark inoculation test was performed using one‐year‐old seedlings from ten different half‐sib lines of narrow‐leaved ash (Fraxinus angustifolia) to examine the variability of host susceptibility to the ash dieback pathogen Hymenoscyphus fraxineus. Sizes of necrotic lesions, production of epicormic shoots and mortality was recorded in all inoculated seedlings 76 days after inoculation. Mortality of different half‐sib lines ranged between 13.3% and 80%. Only one of the tested half‐sib lines showed necrotic lesion sizes, production of epicormic sprouts and percentage of mortality substantially lower than all the other nine half‐sib‐lines of the respective symptom traits. About 20% of half‐sib lines had reduced only one of these three symptoms. The other 70% of tested half‐sib lines had large necrotic lesions, abundant occurrence of epicormic sprouts and high percentage of mortality. Mortality was also significantly influenced by seedling height at the time of inoculation with taller seedlings developing mortality more slowly than smaller seedlings. This study demonstrates significant differences in the susceptibility to H. fraxineus between different half‐sib lines of F. angustifolia and the influence of seedling height on the development of ash dieback.