Leaf Assay Research Articles

Botryosphaeriaceae Infections on Prunus Germplasm: Evaluation of Pathogenicity, Resistance Loci, and Detached Assays of Southeastern United States Isolates

Research of peach fungal gummosis (PFG), which is a vascular disease caused by fungi in the Botryosphaeriaceae family, is particularly important in the southeastern United States, southern China, South Africa, and Western Australia. This disease has been a significant concern for the peach industry in the southeastern United States since the 1970s. The changing climatic conditions in the prunus production areas of the world have worsened the severity of PFG disease. A study of the morphological and genetic profiles of Botryosphaeriaceae species from diseased peach trees found four species in Georgia, Florida, Alabama, and South Carolina. Botryosphaeria dothidea, Lasiodiplodia theobromae, Diplodia seriata, and Neofusicocoum parvum were isolated and identified using conidiospore characters and rDNA sequences of internal transcribed spacer regions and elongation factor α-1 genes. B. dothidea was the most common species from diseased peach trees. A significant implication of this research is the potential resistance in Prunus germplasm, which was genotyped for the resistance locus Botd8 and evaluated for relative susceptibility to one isolate each from B. dothidea, L. theobromae, and D. seriata. Pathogenicity evaluations of peach, almond, and interspecific hybrids used detached stem and leaf assays for susceptibility to PFG based on lesion lengths and gumming scores. L. theobromae inoculation resulted in the most extensive lesions on stems and leaves. Detached stem and leaf assays indicated that Prunus with the Botd8 locus significantly differed in lesion size when infected with B. dothidea and D. seriata. However, the resistant locus had no significant effect on the relative susceptibility of Prunus to L. theobromae. Field and detached stem and leaf assays identified almond cultivars Pioneer and Golden State and Peach × Almond hybrids with Botd8 (+) as tolerant to PFG. Correlations between detached assays and field gumming scores suggested that inoculations on detached stems or leaves under controlled conditions can be used to preliminarily screen for resistance to PFG. These detached assays for specific pathogen isolates provide an opportunity for a relatively fast, nondestructive evaluation of Prunus germplasm and are tools that can be used to identify the components of disease resistance.

First Comprehensive Evaluation of Genetic Variability for Bacterial Blight Resistance in Wild Punica granatum L. Populations from the North-Western Himalayas

Identifying and selecting disease-resistant sources from wild germplasm pools is pivotal due to their ecological benefits and cost-effectiveness. This study encompassed the survey and collection of wild pomegranate genotypes alongside commercial cultivars, followed by screening against native pathogens. Additionally, biochemical analyses were conducted to elucidate defense mechanisms in both resistant and susceptible genotypes. Cuttings from selected germplasm across four districts were obtained, grown for one year, and assessed for pathogenicity and biochemical responses. Microscopic and molecular analyses confirmed the pathogen as Xanthomonas axonopodis pv. punicae (Xap), identified by its distinctive morphology and a 1500 bp amplicon along with phylogeny analysis. Germplasm screening via detached and attached leaf assays identified genotypes with minimal disease severity, highlighting SH-14 and SH-16 as highly resistant in the attached leaf assay. Principal component analysis categorized genotypes based on disease parameters, with PC1 explaining approximately 75% of the variability. Agglomerative hierarchical clustering further grouped genotypes into ten clusters, emphasizing clusters IX and X for their significant resistance traits. Biochemical studies revealed that the resistant genotype SH-16 exhibited higher levels of constitutive defense components like chlorophyll and vitamin C, as well as induced defense components such as phenols, flavonoids, and antioxidants, compared to the susceptible cultivar Bhagwa. These findings underscore the critical roles of both constitutive and induced defense mechanisms in conferring pomegranate resistance to Xap. The study's comprehensive approach in evaluating genetic and biochemical resistance provides valuable insights for breeding disease-resistant pomegranate varieties, marking a significant advancement in exploring pomegranate wild relatives from the Himalayan region for their resistance to bacterial blight.

Evaluating the Efficacy of Endophytic Bacteria in Controlling Rice Sheath Blight: In Vitro and In Vivo Studies

Rice production is highly susceptible to various pathogens, including Rhizoctonia solani, Curvularia lunata, and Epicoccum rostratum, which are major threats in Asia. Exploring biological control methods using endophytic bacteria offers promising opportunities to enhance rice resilience against these lethal diseases. Using 16S RNA sequencing, we identified four endophytic isolates of Bacillus spp. from rice roots, stems, and leaves. We evaluated the antagonistic activity of these endophytic bacterial isolates against rice pathogens both in vitro and in vivo. These isolates inhibited the growth of C. lunata by 82%, R. solani by 79%, and E. rostratum by 88% in vitro. The detached leaf assay for sheath blight (ShB) disease severity in strains ranged from 10.4% to 73.3%. In vivo results showed that B. amyloliquefaciens (R-19) exhibited the lowest disease intensity at 7.2% and the highest disease suppression at 78.8%. The fungicide propiconazole at 0.1% treatment showed the lowest disease intensity of 7.7% and the highest disease suppression of 73.4%, compared to the infected control. Besides biocontrol efficacy, endophytic isolates enhance plant growth parameters, including shoot height, root length, fresh and dry weights, number of tillers, and grains per tiller. Plant hormones abscisic acid (ABA) and gibberellic acids (GA3) increased by 35% and 53%, respectively, due to B. subtilis (R-20) and B. amyloliquefaciens (R-19), while flavonoid and indole acetic acid (IAA) concentrations surged by 30%–80%. Similarly, chlorophyll (a, b), carotenoids, antioxidant enzymatic activity, phenolic content, carbohydrates, and proline contents were higher compared to the control. This study provides a foundation for future studies on novel and eco-friendly biocontrol agents. In addition, our study recommends the integration of endophytic bacteria into sustainable agriculture for enhancing rice production and reducing disease impacts.

Phoma herbarum: A Potential Biocontrol Agent Against Weeds, that Promotes Wheat Growth

The usage of chemical weedicide adversely affects the soil fertility and environment. Hence, in order to reduce the use of chemical weedicide, current study was aimed to isolate plant pathogenic microorganisms from diseased weeds and evaluate their potential as a bioherbicide in wheat field. Twelve bacterial and thirty-one fungal isolates were screened to determine their bioherbicidal activity against prevalent weeds (Avena fatua, Phalaris minor, and Chenopodium album) by using detached leaf assay and in-vitro seed testing methods. Among the forty-three isolates, two potential isolates were selected for further studies. Potential fungal isolates DGL 8C and DGL 7A with significant bioherbicidal activity were molecularly (ITS sequencing) identified as Phoma herbarum R21 (GenBank ID- ON705696) and K_NESO2 (GenBank ID- ON705704). Phoma herbarum R21 was chosen for further research due to its superior herbicidal effect and positive influence on wheat growth. Effective herbicidal activity (up to 90%) of potential isolate was obtained in pre-germination testing, compared to control. Cell free culture filtrate (CFCF) treatment showed nonspecific inhibition in the germination of weeds and wheat. While, CFCF selectively deteriorated the target weeds in post-germination treatment. Phoma herbarum R21 enhanced the growth of Durum wheat varieties Poshan and Tejas, as it promoted the growth of shoot, root, and fresh weight up to 88% compared to control. Phoma herbarum R21 significantly inhibited the growth of phytopathogenic fungi up to 57%. In this study, Phoma herbarum R21 was identified as a potential bioherbicide against the weeds of wheat along with its growth promoting and antifungal activities.

Foliar Resistance to a Natural Population of Plasmopara viticola in Some Grapevine Varieties Grown in Türkiye

ABSTRACTIt is critical to use a population of the pathogen that contains different strains to determine the resistance of grapevine varieties to the downy mildew pathogen Plasmopara viticola in a specific region. This study tested six local grape varieties (Çavuş, Kalecik Karası, Müşküle, Papaz Karası, Sultana and Yapıncak), eight Turkish registered varieties (Barış, Bozbey, Güzgülü, Özer Karası, Reçel Üzümü, Tekirdağ Çekirdeksizi, Trakya İlkeren and Yalova İncisi) and seven varieties of foreign origin (Cabernet Sauvignon, Cardinal, Chardonnay, Cinsault, Isabella, Semillon and Gamay) using detached leaf and leaf disc assays with pathogen populations collected from vineyards. There was a significant correlation for disease severity between the two assay methods. No sporulation was observed on the varieties Çavuş and Isabella in both assay methods. The variety Yapıncak had very low disease severity (2.00%) in the leaf disc assay, and the pathogen did not sporulate on this variety in the detached leaf assay. Cluster analysis was performed to determine the resistance level of the varieties against the pathogen, as different disease severities were observed in some varieties depending on the test method used, and the 21 varieties were classified into four clusters. Barış, Bozbey, Cardinal, Çavuş, Isabella, Semillon and Yapıncak in Cluster I exhibited the lowest disease severity mean (2.88%), while those in Cluster 3, including Cabernet Sauvignon, Güzgülü, Papaz Karası and Yalova Incisi, had the highest disease severity mean (56.75%). These varieties in Clusters 1 and 3 were considered highly resistant and highly susceptible, respectively. Leaf hair density, as reported in official variety descriptions, did not associate with reduced disease severity in the two assays. This study assessed the response of grapevine varieties to the P. viticola population in the region for the first time. The highly resistant and resistant varieties identified will provide new material for breeders and contribute to the improvement of organic viticulture in the area.

First report of Fusarium luffae causing soybean wilt in the USA.

During the 2023 soybean growing season in South Dakota, we scouted a farmer's field and observed soybean (Glycine max (L.) Merr.) plants with wilting symptoms and blighted leaves. Symptomatic stems and leaves were collected from the field to identify associated pathogens. 0.5 cm2 size leaf and stem segments of the sample were surface sterilized by rinsing with 10% bleach for 5 minutes then dipping in 70% ethanol for one minute, and later placing in deionized sterile water for one minute. The sterilized segments were placed on wet filter paper and incubated under fluorescent light for three days. Fungal growth was observed, and the growing mycelia were transferred to potato dextrose agar plates amended with 50 µg/ml Ampicillin (PDAa). Pure culture of the isolate was obtained using single sporing and transferring on new PDAa plates. A dense aerial mycelial growth showing waxy yellow color with a pale orange tinge on the rear side covered the full plate after seven days of incubation at room temperature under fluorescent lights (Figure S1a and b). Developing macroconidia were falcate, curved, smooth to slightly rough, and hyaline with three-five septa (Figure S1c). For molecular identification, DNA of the recovered isolate was extracted and subjected to multiloci PCR (O'Donnell et al., 2010) to amplify and Sanger sequence the internal transcribed spacers region (ITS) (GenBank accession number PP393518), calmodulin (CAM-PP401978), RNA polymerase II second largest subunit (RPB2-PP401980), and translation elongation factor 1-α gene (TEF1-PP401979). The South Dakota isolate (SLSDF2) was identified as Fusairum luffae on NCBI and Fusarioid polyphasic identification databases with 99.40% similarity to Fusarium luffae strain NRRL31167. A phylogeny was inferred based on concatenated TEF1, RPB2, and CAM sequences to show species relatedness (Figure S3). The characterized isolate SDSLF2 was evaluated for soybean pathogenicity using spray inoculations on detached leaves and V2 stage soybean plants (Figure S2a and b). The conidial suspension was prepared by growing the pathogen on mung bean agar for seven days. 2 ml of conidial suspensions (2.6 × 104 conidia/ml) and mock control (sterilized water with 0.1% Tween-20) was sprayed on the detached leaves and whole plants. The experiment was repeated three times with four replicates in each. In the detached leaf assay, leaves were completely blighted (Figure S2a) within 96 hours. In whole plant assays, after two days of incubation, leaf blighting was visible and progressed with time. Four days post-inoculation, the infected plants showed extensive leaf symptoms, and ultimately defoliation occurred (Figure S2b). No symptoms were observed in mock controls of either of the experiments. The pathogen was reisolated from the infected tissues and its identity was confirmed as F. luffae by CAM sequencing fulfilling Koch's postulates. F. luffae has been reported to associated with soybeans in China (Zhao et al., 2022), however, to our knowledge, this is the first report of F. luffae pathogenic on soybeans in the USA, stressing the need to identify resistance sources to avoid any potential disease epidemic.

Exploring the efficacy of endophytic Diaporthe caatingaensis as a biocontrol agent targeting Fusarium strains afflicting coffee plants in Saudi Arabia

BackgroundThe coffee plant is a strategic crop in Saudi Arabia that makes a substantial contribution to the country’s economy. Therefore, it is crucial to continuously monitor and control the fungal phytopathogens that affect coffee plants in order to minimize crop losses and ensure sustainable cultivation. This is the first surveillance report of Fusarium phytopathogens associated with coffee plants in Saudi Arabia. Moreover, the antagonistic efficiency of the endophytic fungus, Diaporthe caatingaensis, was evaluated against the isolated Fusarium phytopathogens. MethodsThe isolated strains were preliminary identified using cultural and microscopic methods and the identification was confirmed using internal transcribed spacer (ITS) sequencing technique. The detached leaf assay was conducted to assess the disease severity of Fusarium phytopathogens against detached coffee leaves. Moreover, dual culture assay was utilized to assess the antagonistic activity of D. caatingaensis. Results and conclusionFusarium oxysporum was found to be the most frequent isolated strain followed by F. solani, F. proliferatum and F. verticillioides strains. Fusarium proliferatum strain was found to be the most severe strain whereas F. solani strain showed the lowest disease severity. On the other hand, propiconazole fungicide was tested for its efficiency against Fusarium pathogens using food poisoning technique, showing that F. oxysporum strain 1 of accession number OP955665 was the most sensitive strain. However, F. proliferatum, F. oxysporum st. 2 (OP959851) and F. solani strains showed no significant response when the propiconazole concentration increase from 150 to 200 ppm. Scanning electron microscope proved the potent antagonistic activity of D. caatingaensis against F. proliferatum and F. oxysporum OP959874 strains through mycoparasitic modes of action as coiling, appressorium formation resulting in complete lysis of fungal mycelium. Accordingly, the current investigation provides the first surveillance data about Fusarium strains associated with coffee plants and also the utilization of D. caatingaensis as a potential biocontrol agent for effective management of Fusarium phytopathogens, avoiding the incidence of fungal resistance to fungicides and the harmful effects of commercial fungicides for sustainable cultivation of coffee in Saudi Arabia.

Isolation and identification of Cladosporium tenuissimum causing leaf blight on garden pea (Pisum sativum L.) in Telangana from southern India

Garden pea (Pisum sativum L.), is an important legume grown worldwide as a source of proteins and carbohydrates. Since pea crop is cultivated globally in cool temperate climatic conditions, increasing temperatures accompanied by high humidity favors fungal infections. A fungal infection causing leaf blight symptoms was frequently observed on the leaves of pea plants grown in greenhouse and field conditions. The blight symptoms showed a water-soaked and wilting appearance with curly leaf edges accompanied by greyish mycelial growth and chlorotic lesions. The disease incidence in field conditions ranged between 5 and 10 %, whereas in the controlled greenhouse conditions, it ranged from 40 to 45%. Two isolates of the fungus, Gp03 and Gp04 were isolated from Arkel pea leaves and purified from a single conidium each. According to Koch's postulates, the pathogenicity tests with these isolates using detached leaf assay and whole plant leaf assays confirmed the pathogen. Morphological and molecular analysis of nucleotide sequences of the D1-D2 region, translation elongation factor-1α (TEF) and actin (ACT) genes confirmed these isolates as members of the genus, Cladosporium. The phylogenetic relatedness of these isolates with other members of the Cladosporium genus revealed them as fungal strains of Cladosporium tenuissimum. Further, these isolates were also confirmed as C. tenuissimum by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh in India, and provided the accession nos. MTCC 13581 and MTCC 13582 to the C. tenuissimum strains, Gp03 and Gp04 respectively. This is the first report of Cladosporium tenuissimum infecting garden pea in Telangana, India.

Assessing Soybean Cultivar Resistance to Target Spot Using a Detached Leaf Assay

Target spot (TS), caused by the fungus Corynespora cassiicola, is a foliar disease of increasing importance to soybean in the US. Identifying cultivar resistance to TS is challenging. The objective of this research was to determine the reactions of a diverse collection of soybean genotypes to target spot using a detached leaf inoculation method. A detached unifoliate leaf of each soybean genotype was placed in a Petri dish on moist filter paper, wounded at three locations, inoculated with 10 μl, of a 105 conidial suspension of C. cassiicola, and incubated at room temperature (23°C). The percent leaf area with TS symptoms (TSS) was measured digitally 7, 10, 14, and 17 days after inoculation (DAI) and the area under the disease progress curve (AUDPC) calculated. Thirty-seven soybean genotypes and three soybean cultivars were evaluated in three replicated trials. At 17 DAI, target spot symptoms ranged from 2 to 84% TSS. Significant differences occurred between soybean genotypes at each rating time in each trial. Across the three trials, the reaction of most soybean genotypes ranged from resistant to susceptible. These soybean genotypes were evaluated in the field with natural inoculum at two locations in 2022. Target spot developed late but differed significantly between genotypes averaging from 0.35 to 3.35% TSS. Field results were not significantly correlated to the detached leaf results. The detached leaf assay is another tool for screening soybean genotypes for resistance to TS but needs field validation.

Genome-wide search and gene expression studies reveal candidate effectors with a role in pathogenicity and virulence in Fusarium graminearum

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) disease in wheat worldwide. Although F. graminearum is reported to secrete several effectors, their role in virulence and pathogenicity is unknown. The study aimed at identifying candidate genes with a role in pathogenicity and virulence using two different host systems, Arabidopsis thaliana and wheat, challenged with F. graminearum TN01. Detached leaf assay and histological studies revealed the virulent nature of TN01. A genome-wide in silico search revealed several candidate genes, of which 23 genes were selected based on reproducibility. Gene expression studies by reverse transcriptase–polymerase chain reaction (RT-PCR) in leaf tissues of Arabidopsis and the two wheat genotypes, the susceptible (Sonalika) and the resistant (Nobeoka Bozu/Nobeoka), compared with mock-treated controls in a time-course study using fungal- and plant-specific genes as internal controls revealed that these genes were differentially regulated. Further, expression of these candidates in F. graminearum–inoculated Sonalika and Nobeoka spikes compared with mock-treated controls revealed their role in pathogenicity and virulence. Gene ontology studies revealed that some of these secretory proteins possessed a role in apoptosis and ceratoplatanin and KP4 killer toxin syntheses. A three-dimensional protein configuration was performed by homology modeling using trRosetta. Further, real-time quantitative PCR (RT-qPCR) studies in F. graminearum–inoculated Arabidopsis and wheat at early time points of inoculation revealed an increased expression of the majority of these genes in Sonalika, suggesting their possible role in pathogenicity, whereas low mRNA abundance was observed for 11 of these genes in the resistant genotype, Nobeoka, compared with Sonalika, indicating their role in virulence of F. graminearum.

Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability

The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Evaluating the Biocontrol Efficacy and Antioxidant Potential of Phellinus caribaeo-quercicola-A First Report Dual-Action Endophyte From Inula racemosa Hook. F.

Phellinus caribaeo-quercicola is a basidiomycetous fungus, isolated as an endophyte in this study from the healthy and symptomless leaves of Inula racemosa Hook. f., an important medicinal herb growing in Kashmir Himalaya. This study combines morphological, molecular and phylogenetic techniques to identify the fungal endophyte, using the ITS sequence of nrDNA. A detached leaf assay was conducted to assess the pathogenicity of the fungal endophyte suggesting its mutually symbiotic relationship with the host. The authors also investigated the antifungal potential of the isolated endophytic strain to ascertain its use as a biocontrol agent. The study shows that P. caribaeo-quercicola INL3-2 strain exhibits biocontrol activity against four key fungal phytopathogens that cause significant agronomic and economic losses: Aspergillus flavus, Aspergillus niger, Fusarium solani, and Fusarium oxysporum. Notably, P. caribaeo-quercicola INL3-2 strain is highly effective against A. flavus, with an inhibition percentage of 57.63%. In addition, this study investigates the antioxidant activity of P. caribaeo-quercicola INL3-2 strain crude extracts using ethyl acetate and methanol as solvents. The results showed that the methanolic fraction of P. caribaeo-quercicola exhibits potential as an antioxidant agent, with an IC50 value of 171.90 ± 1.15 µg/mL. This investigation is first of its kind and marks the initial report of this fungal basidiomycete, P. caribaeo-quercicola, as an endophyte associated with a medicinal plant. The findings of this study highlight the potential of P. caribaeo-quercicola INL3-2 strain as a dual-action agent with both biocontrol and antioxidant properties consistent with the medicinal properties of Inula racemosa. This endophytic fungus could be a promising source of natural compounds for use in agriculture, medicine, and beyond.

Enhanced antifungal activity of sugarcane cv. NCo310 expressing chimeric chitinase 42

The development of sustainable and environmentally safe strategies for crop protection against fungal pathogens is a significant challenge in agriculture. This paper discusses the successful transfer of a chimeric chitinase 42 gene into sugarcane using the biolistic method, resulting in an enhanced antifungal activity. Chitinases possess the enzymatic capability to degrade chitin, a key component of the cell walls in various fungal phytopathogens. In this research, we employed a chimeric chitinase 42 that combines the 42 kDa chitinase from Trichoderma atroviridae with a chitin binding domain from Serratia marcescens chitinase B. This chimeric chitinase exhibits higher antifungal activity against phytopathogenic fungi. PCR analysis confirmed the integration of the transgene into the genome of transformants, while the expression of the transgene was assessed using reverse transcription-PCR. Transgenic lines showed to have different chitinase activity and a high increase in chitinase activity was detected in lines T3, T5 and T6 with about 18 U/mg chitinase activity compared to the control plants (2.8 U/mg). Antifungal activity of transgenic lines T3, T5 and T6 on sugarcane fungal pathogens was shown in radial diffusion and detached leaf assays. The enhanced antifungal activity observed in the transformed sugarcane lines indicates the potential of chimeric chitinase 42 as an effective antifungal agent.

Potato spindle tuber viroid (PSTVd) loop 27 mutants promote cell-to-cell movement and phloem unloading of the wild type: Insights into RNA-based viroid interactions

Variations in infection progression with concurrent or prior infections by different viruses, viroids, or their strains are evident, but detailed investigations into viroid variant interactions are lacking. We studied potato spindle tuber viroid intermediate strain (PSTVd-I) to explore variant interactions. Two mutants, U177A/A182U (AU, replication- and trafficking-competent) and U178G/U179G (GG, replication-competent but trafficking-defective) on loop 27 increased cell-to-cell movement of wild-type (WT) PSTVd without affecting replication. In mixed infection assays, both mutants accelerated WT phloem unloading, while only AU promoted it in separate leaf assays, suggesting that enhancement of WT infection is not due to systemic signals. The mutants likely enhance WT infection due to their loop-specific functions, as evidenced by the lack of impact on WT infection seen with the distantly located G347U (UU) mutant. This study provides the first comprehensive analysis of viroid variant interactions, highlighting the prolonged phloem unloading process as a significant barrier to systemic spread.

Target and non-target effects of insecticide use during ornamental milkweed production.

There are widespread public efforts to conserve wildlife in urbanized landscapes via the installation of nursery-grown plants that support Lepidoptera taxa. Insecticides are commonly used during nursery production to suppress key plant pests, and many products have extended periods of toxicity and affect a wide range of herbivore taxa. While there are plentiful toxicological data on bee species, predominantly the Western honey bee (Apis mellifera L.), little is known about how insecticides affect nonpest lepidopterans. Lepidoptera has different modes of exposure (e.g., leaf-feeding) and differences in susceptibility to insecticide target sites compared to bees. Consequently, many products compatible with bee conservation pose an uncertain risk to nonpest lepidopterans and thus may represent an under-recognized conflict with conservation efforts. Using the monarch butterfly (Danaus plexippus, L.), tropical milkweed (Asclepias curassavica, L.), and oleander aphid (Aphis nerii, Fonscolombe, 1841) system, we conducted leaf and whole-plant feeding assays to evaluate effects of acute and chronic monarch exposure to industry standard and alternative reduced-risk insecticides used during nursery production. We also evaluated the efficacy of these insecticides against their target pest, the oleander aphid. Our results indicate that insecticides used to control pests on ornamental milkweed can cause monarch larval mortality up to 4 wk after treatment application. Furthermore, the duration of aphid suppression is often shorter than the duration of adverse effects on monarchs. This study demonstrates a conflict between insect pest management and Lepidoptera conservation during ornamental plant production and has implications for the conservation value of ornamentals after retail sale.

Synthesis, bioactivity and molecular docking of novel coumarin-quinolinamide containing monocyclic monoterpenes as potential SDH inhibitors

Succinate dehydrogenase inhibitors (SDHI) are widely used in plant antifungal agents. To break the barrier of resistance, a series of new coumarin-quinolinamide SDHI derivatives containing monocyclic monoterpenes were designed and synthesized using natural camphor or natural menthol as lead compounds, and their structures were characterized by FT-IR, 1H NMR, 13C NMR and HRMS. In vitro antifungal activity test results showed that the target compounds had inhibitory activity against most plant pathogenic fungi, especially against Rhizoctonia solani and Phytophthora nicotianae var. nicotianae, which was superior to other fungi. Among them, compounds 11t and 12t showed better inhibitory effects on Rhizoctonia solani than the commercial fungicide tricyclazole, with EC50 of 16.90 mg/L and 27.09 mg/L, respectively. Based on this, the detached leaf assay was used to further explore the in vivo therapeutic and preventive effects of compounds 11t and 12t on rice sheath blight in plants. The results of the antifungal activity test in vivo showed that compound 11t was slightly better than the positive control, while compound 12t was close to the control effect of azoxystrobin. More importantly, the more bioactive compounds 11t and 12t did not affect the germination of cowpea seeds and also did not affect the growth of normal human hepatocytes and kidney cells. Molecular docking experiments showed that the introduction of the diethylamino group could promote the formation of hydrogen bonds between the hydrogen atom on the amide bond of the target compound and residue Tyr91, and compounds 11t and 12t had smaller free energy between the amino acid residue, which would enhance the antifungal activity of the compounds. These studies proved the potential value of this series of compounds for the study of novel amide-based SDHI fungicides.

Exploring Potential Surrogate Systems for Studying the Early Steps of the Sporisorium scitamineum Pathogenesis.

Despite its global importance as a primary source of table sugar and bioethanol, sugarcane faces a significant threat to its production due to diseases. One of these diseases, sugarcane smut, involves the emergence of a whip-like structure from the host apical shoot. The slow onset of this pathogenesis is the most substantial challenge for researchers to investigate the molecular events leading to resistance or susceptibility. In this study, we explored the early interaction between the smut fungus Sporisorium scitamineum and foliar tissues of the model plants Arabidopsis thaliana and Nicotiana benthamiana. Upon inoculation with the fungus, A. thaliana showed a compatible reaction, producing lesions during fungus colonization, whereas N. benthamiana showed signs of nonhost resistance. In addition, we propose a sugarcane detached leaf assay using plants cultivated in vitro to reveal sugarcane smut response outcomes. We used two sugarcane genotypes with known contrasting reactions to smut in the field. Although there is no evidence of sugarcane smut fungus infecting host leaves naturally, the sugarcane detached leaf assay enabled a rapid assessment of disease outcomes. Different symptoms in the detached leaves after inoculation distinguished smut-susceptible and smut-resistant sugarcane genotypes. Microscopic observations and gene expression analysis of S. scitamineum candidate effectors confirmed the fungal growth and its restriction on the compatible and incompatible interactions, respectively. These findings offer new prospects into the disease phenotyping of S. scitamineum, which could greatly expedite the comprehension of the initial stages of the pathogenesis and predict smut resistance in sugarcane genotypes.

Design, Synthesis, and 3D-QASR of 2-Ar-1,2,3-triazole Derivatives Containing Hydrazide as Potential Fungicides.

A series of novel 2-Ar-1,2,3-triazole derivatives were designed and synthesized based on our previously discovered active compound 6d against Rhizoctonia solani. Most of these compounds exhibited good antifungal activity against R. solani at a concentration of 25 μg/mL. Based on the results of biological activity, we established a three-dimensional quantitative structure-activity relationship (3D-QSAR) model that guided the synthesis of compound 7y. Compound 7y exhibited superior activity against R. solani (EC50 = 0.47 μg/mL) compared to the positive controls hymexazol (EC50 = 12.80 μg/mL) and tebuconazole (EC50 = 0.87 μg/mL). Furthermore, compound 7y demonstrated better protective activity than the aforementioned two commercial fungicides in both detached leaf assays and greenhouse experiments, achieving 56.21% and 65.75% protective efficacy, respectively, at a concentration of 100 μg/mL. The ergosterol content was determined and molecular docking was performed to explore the mechanism of these active molecules. DFT calculation and MEP analysis were performed to illustrate the results of this study. These results suggest that compound 7y could serve as a novel 2-Ar-1,2,3-triazole lead compound for controlling R. solani.

Effective Control of Sclerotinia Stem Rot in Canola Plants Through Application of Exogenous Hairpin RNA of Multiple Sclerotinia sclerotiorum Genes.

Sclerotinia stem rot is a globally destructive plant disease caused by Sclerotinia sclerotiorum. Current management of Sclerotinia stem rot primarily relies on chemical fungicides and crop rotation, raising environmental concerns. In this study, we developed an eco-friendly RNA bio-fungicide targeting S. sclerotiorum. Six S. sclerotiorum genes were selected for double-stranded RNA (dsRNA) synthesis. Four genes, a chitin-binding domain, mitogen-activated protein kinase, oxaloacetate acetylhydrolase, and abhydrolase-3, were combined to express hairpin RNA in Escherichia coli HT115. The effect of application of total RNA extracted fromE. coliHT115 expressing hairpin RNA on disease progressive and necrosis lesions was evaluated. Gene expression analysis using real-time PCR showed silencing of the target genes using 5 ng/µl of dsRNA in a fungal liquid culture. A detached leaf assay and greenhouse application of dsRNA on canola stem and leaves showed variation in the reduction of necrosis symptoms by dsRNA of different genes, with abhydrolase-3 being the most effective. The dsRNA from a combination of four genes reduced disease severity significantly (P = 0.01). Plants sprayed with hairpin RNA from four genes had lesions that were almost 30% smaller than those of plants treated with abhydrolase-3 alone, in lab and greenhouse assays. The results of this study highlight the potential of RNA interference to manage diseases caused by S. sclerotiorum; however, additional research is necessary to optimize its efficacy.