Inoculation Assays Research Articles

Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue

Beneficial soil microorganisms, particularly plant probiotic bacteria (PPB), play a pivotal role in promoting plant growth, development, and overall health through root colonization. PPB-based biofertilizers offer a sustainable and eco-friendly alternative to conventional agricultural inputs. This study evaluates the plant probiotic potential of three native bacterial strains Rhizobium sp. ACO-34A, Sinorhizobium mexicanum ITTG R7T, and Sinorhizobium chiapasense ITTG S70T to enhance the growth, quality, and sugar content of Agave tequilana. A comprehensive genomic and functional analysis was conducted for each strain to assess their plant probiotic traits. Additionally, a greenhouse inoculation assay was performed on six-month-old agave seedlings at the “piña” stage to evaluate the effects of these strains on plant growth and sugar content. Comparative genomic analysis revealed that these rhizobial strains harbor genes associated with key plant probiotic traits, reinforcing their role in enhancing plant development. The results demonstrated significant effects (p < 0.05) on growth and sugar content in inoculated plants. ACO-34A increased plant height by 35.4%, fresh weight by 41.5%, and inulin content by 57.3%, while ITTG-R7T showed improvements of 26.4%, 35.2%, and 38.2%, respectively, compared to the control, and ITTG S70T also exhibited enhancements, although to a lesser extent, with increases of 23.5% in plant height, 28.9% in fresh weight, and 31.2% in inulin content. These findings highlight the biofertilizer potential of these native rhizobial strains, particularly Rhizobium sp. ACO-34A, positioning them as promising candidates for the sustainable cultivation of A. tequilana.

A typical NLR recognizes a family of structurally conserved effectors to confer plant resistance against adapted and non-adapted Phytophthora pathogens.

Plants possess remarkably durable resistance against non-adapted pathogens in nature. However, the molecular mechanisms underlying this resistance remain poorly understood, and it is unclear how the resistance is maintained without coevolution between hosts and the non-adapted pathogens. In this study, we used Phytophthora sojae (Ps), a non-adapted pathogen of N. benthamiana (Nb), as a model and identified an RXLR effector that determines Nb incompatibility to Ps. Knockout of this RXLR effector in Ps enables successful infection of Nb, leading us to name it AvrNb (Avirulence gene in Nb). A systematic screening of Nb NLR genes further revealed that NbPrf, previously reported to be a receptor of bacterial avirulence proteins, is the NLR protein responsible for mediating AvrNb recognition and initiating the hypersensitive response (HR). Mutation of NbPrf completely makes Nb compatible to Ps. We found that AvrNb is conserved among multiply Phytophthora pathogens, and these homologs also induce NbPrf-dependent HR. Remarkably, further inoculation assay showed that NbPrf is involved in plant immunity to two adapted Phytophthora pathogens, P. infestans and P. capsici. Our findings suggest that NbPrf represents a promising breeding resource for resistance to Phytophthora pathogens, and also demonstrate that the conserved effectors present in both adapted and non-adapted pathogens may provide sufficient selective pressure to maintain the remarkably durable incompatibility between plants and non-adapted pathogens.

Enhanced Resistance to Pokkah Boeng Disease in Sugarcane Through Host-Induced Gene Silencing Targeting FsCYP51 in Fusarium sacchari.

Pokkah boeng disease (PBD), a common and highly destructive disease of sugarcane, is mainly caused by Fusarium sacchari. Breeding sugarcane resistant to PBD is challenging due to the limited availability of immune or highly resistant germplasm resources. Host-induced gene silencing (HIGS) based on RNA interference (RNAi) is a promising disease-control method that offers strong disease-targeting ability with low environmental impact. This study found that silencing either three FsCYP51 genes (FsCYP51A, FsCYP51B and FsCYP51C) simultaneous or two of them (FsCYP51A and FsCYP51C) could inhibit the growth, development, and virulence of F. sacchari. Subsequently, we developed CYP51-HIGS transgenic sugarcane lines using gene-gun genetic transformation and obtained seven lines expressing dsFsCYP51. Both the results of laboratory inoculation assays and field trials indicated that all the seven transgenic lines had significant resistance to PBD. Moreover, in the field trials, the yield losses of transgenic sugarcane due to PBD were reduced compared with those of the control. This is the first report using the HIGS strategy to inhibit PBD infection in sugarcane. This breakthrough provides clear guidelines and practical approaches for the future breeding of sugarcane varieties with strong antifungal resistance.

Unveiling of Refined Isolation, Growth Conditions and Inoculation Assay for Ephelis japonica Inciting Udbatta Disease in Rice

Udbatta incited by Ephelis oryzae Syd is a minor significant seed borne, sporadic and systemic disease in rice. Isolation of this pathogen is challenging due to its weak growth and the prevalence of other rapidly proliferating fungi like Curvularia, Aspergillus etc. In this study, a streamlined spore-drop technique was standardized for rapid isolation of Ephelis japonica from infected rice panicles. This method achieved pure cultures with minimal contamination (7.50%) in the shortest time (25 days) with a growth rate of 1.7 mm/day, compared to the spore dilution method (32.50%) in 65 days with 0.83 mm/day and standard tissue isolation method (50.25%) in 70 days with 0.45 mm/day. Optimal conditions for fungal growth in axenic culture were also standardized. E. japonica recorded the fastest growth on potato dextrose agar (PDA) medium at an incubation temperature of 27°C under dark conditions, which were more conducive to growth than light incubation. The fungus recorded a maximum radial growth of 35.60 mm in 25 days with growth rate of 1.42 mm/day on PDA supplemented with 4% dextrose as compared to 30.27 mm and 1.21 mm/day in 2% dextrose. Furthermore, an inoculation method was standardized for inducing Udbatta disease by treating rice seeds with a fungal suspension for 1–2 hours, followed by overnight drying and sowing resulted in disease incidence of 6.25 % and no disease was observed in stem inoculation and rice head inoculation. Plants grown from seeds treated with fungal suspension exhibited characteristic symptoms like black, erect, cylindrical spike-like structured panicles with grains matted with fungal mat. This refined isolation technique, with optimized culture conditions, provides a robust framework for advancing research on E. japonica and Udbatta disease in India.

OsbHLH5 Synergically Regulates Phenolamide and Diterpenoid Phytoalexins Involved in the Defense of Rice Against Pathogens.

Rice (Oryza sativa) produces phenolamides and diterpenoids as major phytoalexins. Although the biosynthetic pathways of phenolamides and diterpenoids in plants have been revealed, knowledge of their accumulation regulatory mechanisms remains limited, and, in particular, no co-regulatory factor has been identified to date. Here, using a combined co-expression and evolutionary analysis, we identified the basic helix-loop-helix (bHLH) transcription factor OsbHLH5 as a positive bifunctional regulator of phenolamide and diterpenoid biosynthesis in rice. Metabolomic analysis revealed that OsbHLH5 significantly increased the content of phenolamides (such as feruloyl tryptamine (Fer-Trm) and p-coumaroyl tyramine (Cou-Tyr)) and diterpenoid phytoalexins (such as momilactones A, momilactones B) in the overexpression lines, while their content was reduced in the OsbHLH5 knockout lines. Gene expression and dual-luciferase assays revealed that OsbHLH5 activates phenolamide biosynthetic genes (including putrescine hydroxycinnamoyltransferase 3 (OsPHT3), tyramine hydroxycinnamoyltransferases 1/2 (OsTHT1/2), and tryptamine benzoyltransferase 2 (OsTBT2)) as well as diterpenoid biosynthetic genes (including copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4/7/10/11 (OsKSL4/7/10/11)). Furthermore, we have demonstrated that OsbHLH5 is induced by jasmonic acid (JA), while pathogen inoculation assays indicated that the overexpression of OsbHLH5 in transgenic rice plants leads to enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). Overall, we have identified a positive regulator of phenolamide and diterpenoid biosynthesis and have demonstrated that biotic stress induces phytoalexin accumulation partly in an OsbHLH5-dependent manner, providing new insights into the metabolic interactions involved in pathogen response and offering valuable gene resources for the development, through genetic improvement, of new rice varieties that are resistant to diseases.

Emerging Begomovirus ToLCNDV Molecular Detection & Chip Grafting Inoculation Transmission Assay under Natural Conditions in Tomato Plants from District Poonch Azad Kashmir & Pakistan

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite, emerging Begomovirus species, usually coupled with satellite molecules and affects tomato cultivation in entire Pakistan. Present study was conducted to evaluate and analyze tomato germplasm against ToLCNDV using Chip graft inoculation assay (CGIA) as most accurate and reliable technique. Molecular studies were conducted for confirmation. According to PCR results, viral contamination was confirmed as ToLCNDV was recorded from all tested seedling samples. Further mechanical transmission confirmed that seedlings from ToLCNDV-infected seeds act as inoculum. Based on disease symptoms developed, plants were classified as tolerant, moderately tolerant, moderately susceptible, and susceptible. It is pertinent to mention that the magnitude of the virus impact particularly on hosts different from Solanaceae and Cucurbitaceae, and seed transmission and ToLCNDV qualify as a potential candidate of Quarantine Pest. Present work is novel study demonstrating ToLCNDV as a seed-transmissible virus in tomato plants from Rawalakot Azad Kashmir & this research further provides a fictitious starting point for plant pathologists suggesting practical implications regarding management approaches.

Discosia brasiliensis causing Discosia leaf blight on tea plant (Camellia sinensis) in China

Tea (Camellia sinensis), a perennial evergreen shrub, is one of the most important cash crops in China. Tea leaves with symptoms of wilt disease was observed in Fengqing County, Lincang City, Yunnan Province, China. Large irregular jujube-red necrotic spots appeared on the leaves of tea plants, and the lesions with grayish white edge were accompanied by a certain degree of shrinkage. In the tea garden planting base, the natural disease incidence reached 40%–50 %, which significantly affects the yield of tea. One putative pathogen was isolated from three symptomatic tea plant leaves and was identified as Discosia brasiliensis using morphology and molecular phylogeny of multi-loci (ITS, LSU, tub, rpb2) sequence data. Using D. brasiliensis strains for artificial inoculation assay on the tea plant leaves, leaf atrophy symptom in leaves which is similar to those observed in the tea planting base, and the putative pathogen was re-isolated to fulfill Koch's postulates. This is the first report of wilt disease caused by Discosia brasiliensis in China.

Screening of potato germplasm for resistance to potato virus y and potato leafroll virus using DAS-ELISA

Potato (Solanum tuberosum L.) ranks as the fourth most important staple food globally, after rice, wheat, and maize. In Pakistan, Potato virus Y (PVY) and Potato leafroll virus (PLRV) are among the most critical pathogens affecting potato crops, causing yield losses of up to 90%. Fifteen advanced potato lines from the Potato Program of the Horticultural Research Institute (HRI) were screened for resistance to these economically significant viruses. The experiment was conducted in the glasshouse of the Crop Diseases Research Institute, NARC, Islamabad, during 2023-24, following a completely randomized design with three replications per clone. For screening, small tubers of fifteen potato clones, including one check variety, were planted in pots within the glasshouse. PVY was mechanically transmitted, while a chip graft inoculation assay was used for screening against PLRV. Symptoms began to appear two weeks after inoculation, and data were recorded four weeks post-inoculation. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was performed on all plants to confirm the presence of both viruses serologically. The findings revealed that the line HRI-P1 remained asymptomatic and was categorized as highly resistant to both viruses, based on symptom observation and ELISA testing. Six clones exhibited varying levels of resistance, three showed moderate susceptibility, one was susceptible, and three were highly susceptible to PVY. The study highlights the availability of valuable sources of resistance within the Potato Program. HRI, NARC, which could be utilized in breeding potential future varieties. The disease-resistant genotypes identified will be instrumental in future potato breeding programs.

Rmg10, a Novel Wheat Blast Resistance Gene Derived from Aegilops tauschii.

Wheat blast, caused by Pyricularia oryzae (syn. Magnaporthe oryzae) pathotype Triticum (MoT), is a devastating disease that can result in up to 100% yield loss in affected fields. To find new resistance genes against wheat blast, we screened 199 accessions of Aegilops tauschii, the D genome progenitor of common wheat (Triticum aestivum), by seedling inoculation assays with Brazilian MoT isolate Br48 and found 14 resistant accessions. A synthetic hexaploid wheat line (Ldn/KU-2097) derived from a cross between the T. turgidum 'Langdon' (Ldn) and resistant A. tauschii accession KU-2097 exhibited resistance in seedlings and spikes against Br48. In an F2 population derived from 'Chinese Spring' × Ldn/KU-2097, resistant and susceptible individuals segregated in a 3:1 ratio, suggesting that the resistance from KU-2097 is controlled by a single dominant gene. We designated this gene Rmg10. Genetic mapping using an F2:3 population from the same cross mapped the RMG10 locus to the short arm of chromosome 2D. Rmg10 was ineffective against Bangladesh isolates but effective against Brazilian isolates. Field tests in Bolivia showed increased spike resistance in a synthetic octaploid wheat line produced from a cross between common wheat cultivar 'Gladius' and KU-2097. These results suggest that Rmg10 would be beneficial in farmers' fields in South America.

Streptomyces pratensis S10 Promotes Wheat Plant Growth and Induces Resistance in Wheat Seedlings against Fusarium graminearum.

Fusarium graminearum, a devastating fungal pathogen, causes great economic losses to crop yields worldwide. The present study investigated the potential of Streptomyces pratensis S10 to alleviate F. graminearum stress in wheat seedlings based on plant growth-promoting and resistance-inducing assays. The bioassays revealed that S10 exhibited multiple plant growth-promoting properties, including the production of siderophores, 1-aminocyclopropane-1-carboxylic acid deaminase (ACC), and indole-3-acetic acid (IAA), phosphate solubilization, and nitrogen fixation. Meanwhile, the pot experiment demonstrated that S10 improved wheat plant development, substantially enhancing wheat height, weight, root activity, and chlorophyll content. Consistently, genome mining identified abundant genes associated with plant growth promotion. S10 induced resistance against F. graminearum in wheat seedlings. The disease incidence and disease index reduced by nearly 52% and 65% in S10 pretreated wheat seedlings, respectively, compared with those infected with F. graminearum only in the non-contact inoculation assay. Moreover, S10 enhanced callose deposition and reactive oxygen species (ROS) accumulation and induced the activities of CAT, SOD, POD, PAL, and PPO. Furthermore, the quantitative real-time PCR (qRT-PCR) results indicated that S10 pretreatment increased the expression of SA- (PR1.1, PR2, PR5, and PAL1) and JA/ET-related genes (PR3, PR4a, PR9, and PDF1.2) in wheat seedlings upon F. graminearum infection. In summary, S. pratensis S10 could be an integrated biological agent and biofertilizer in wheat seedling blight management and plant productivity enhancement.

Biological and Molecular Characterization of the Cucumber Mosaic Virus Infecting Purple Coneflowers in China

Purple coneflower (Echinacea purpurea L.), which is a perennial herbaceous plant belonging to the Asteraceae family, is extensively cultivated because of its medicinal applications. However, in Hohhot, Inner Mongolia, China, purple coneflowers in the field exhibited symptoms such as mottle, mosaic, and crinkle. This study aimed to explore the biological and molecular characteristics of the cucumber mosaic virus (CMV) infecting the purple coneflowers in China. We observed isometric particles approximately 30 nm in diameter in the symptomatic leaf specimens. Infection with the CMV was confirmed via high-throughput sequencing and RT-PCR validation. Mechanical inoculation assays demonstrated that the CMV-SGJ isolate could infect both Nicotiana benthamiana and Nicotiana tabacum. Three viral genomic components were identified: RNA1 with 3321 nucleotides, RNA2 with 3048 nucleotides, and RNA3 with 2209 nucleotides. Phylogenetic analysis revealed that the CMV-SGJ isolate clustered into phylogenetic subgroup IA, exhibiting a nucleotide identity of 92.2–95% with subgroup IA CMV isolates in GenBank. This report is the first documentation of the complete genome of the CMV infecting purple flowers in China.

Plant growth promoting activities of endophytic bacteria from Melia azedarach (Meliaceae) and their influence on plant growth under gnotobiotic conditions

Bacteria that live asymptomatically within plant tissues are known as endophytes. Because of the close relation with the plant host, they have been a matter of interest for application as plant growth promoters. Melia azedarach is a widely distributed medicinal tree with proven insecticidal, antimicrobial, and antiviral activity. The aim of this study was to isolate and characterize endophytic bacteria from M. azedarach and analyze their plant growth promoting activities for the potential application as biological products. Bacteria were isolated from roots and leaves of trees growing in two locations of Northeastern Argentina. The isolates were characterized by repetitive extragenic palindromic sequence PCR and 16S rDNA sequence analysis. The plant growth-promoting activities were assayed in vitro, improvement of plant growth of selected isolates was tested on M. azedarach plantlets, and the effect of selected ACC deaminase producing isolates was tested on tomato seedlings under salt-stress conditions. The highest endophytic bacterial abundance and diversity were obtained from the roots. All isolates had at least one of the assayed plant growth-promoting activities and 80 % of them had antagonistic activity. The most efficient bacteria were Pseudomonas monteilii, Pseudomonas farsensis, Burkholderia sp. and Cupriavidus sp. for phosphate solubilization (2064 μg P ml−1), IAA production (94.7 μg ml−1), siderophore production index (5.5) and ACC deaminase activity (1294 nmol α-ketobutyrate mg−1 h−1). M. azedarach inoculation assays revealed the bacterial growth promotion potential, with Pseudomonas monteilii, Pseudomonas farsensis and Cupriavidus sp. standing out for their effect on leaf area, leaf dry weight, specific leaf area, and total Chl, Mg and N content, with increases of up to 149 %, 58 %, 65 %, 178 %, 76 % and 97.7 %, respectively, compared to NI plants. Efficient ACC deaminase-producing isolates increased stress tolerance of tomato plants under saline condition. Overall, these findings indicate the potential of the endophytic isolates as biostimulant and biocontrol agents.

FaERF2 activates two β-1,3-glucanase genes to enhance strawberry resistance to Botrytis cinerea

Ethylene response factor (ERF) is a class of plant-specific transcription factors that play an important role in plant growth, development, and stress response. However, the underlying mechanism of strawberry ERFs in pathogenic responses against Botrytis cinerea (B. cinerea) remains largely unclear. In this study, we isolated FaERF2, a nucleus-localized ERF transcription factor from Fragaria x ananassa. Transiently overexpressing FaERF2 in strawberry fruits significantly enhances their resistant ability to B. cinerea, while silencing FaERF2 in strawberry fruits enhances their susceptibility to B. cinerea. In addition, we found that FaERF2 could directly bind to the cis-acting element GCC box in the promoters of two β-1,3-glucanase genes, FaBG-1 and FaBG-2, and activate their expression. Finally, both strawberry fruits transient expression followed by B. cinerea inoculation assays and recombinant protein incubation tests collectively substantiated the inhibitory effect of FaBG-1 and FaBG-2 on B. cinerea mycelium growth. These results revealed the molecular regulation mechanism of FaERF2 in response to B. cinerea and laid foundations for creating disease-resistance strawberry cultivar through genome editing approach.

Transcriptional activation of MdDEF30 by MdWRKY75 enhances apple resistance to Cytospora canker

Defensin is an essential component of plant development, and it plays an indispensable role in pathogen resistance. However, the molecular function of defensins under pathological conditions of Cytospora canker has not been characterized in apple plants. In the present study, we present a detail overview of phylogeny and structure of 29 defensins (MdDEF) in apple. Expression analysis revealed that MdDEF genes was spatiotemporally diverse across apple tissues. Five MdDEF genes were found to be significantly up-regulated following a challenge with Cytospora mali. The transgenic overexpression of five defensin genes in apple calli markedly enhanced resistance to C. mali. Among them, MdDEF30 was strongly induced and conferred the highest level of resistance in vivo. Meanwhile, antifungal activity assays in vitro demonstrated that a recombinant protein produced from MdDEF30 could inhibit the growth of C. mali. Notably, MdDEF30 promoted the accumulation of reactive oxygen species (ROS) and activated defense-related genes such as PR4, PR10, CML13 and MPK3. Co-expression regulatory network analysis showed that MdWRKY75 may regulate the expression of MdDEF30. Further yeast one-hybrid (Y1H), luciferase, and chromatin Immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays verified that MdWRKY75 could directly bind to the promoter of MdDEF30. Importantly, pathogen inoculation assays confirmed that MdWRKY75 positively regulates resistance by transcriptionally activating MdDEF30. Taken together, these results demonstrated that MdDEF30 promotes resistance to C. mali in apple plants and that MdWRKY75 regulates MdDEF30 expression during the induction of resistance, thereby clarifying biochemical mechanisms of resistance to C. mali in apple trees.

Comparison of two different experimental environments for resistance screenings for the leafhopper-transmitted wheat dwarf virus in wheat

Wheat dwarf virus (WDV) causes high yield losses in wheat and other cereals and is therefore an important pathogen transmitted by the leafhopper Psammotettix alienus. Climate change will increase infections by insect-transmitted viruses due to the increasing spread of vectors. In the context of integrated pest management, the cultivation of WDV-resistant/tolerant varieties is an effective way of controlling WDV. Evaluation of tolerant/resistant genotypes is based on inoculation with viruliferous leafhoppers and subsequent phenotyping in gauze houses under semi-field conditions. For successful screening, it is important to ensure the uniform and reproducible inoculation of plants. Abiotic conditions, particularly temperature, have a critical influence on inoculation success, and thus, variations in infection rates were observed within and between previous replicates in the field. Furthermore, the leafhopper population reared in the greenhouse has to be reestablished after each infection, which delays the screening process. We addressed these issues by developing an improved inoculation assay in which plants are inoculated in small infection hoods in the greenhouse before being planted out in gauze houses. This procedure allows optimal environmental conditions for WDV infection of test plants and allows the plants with WDV infection to develop under natural environmental conditions for symptom scoring. In addition, the viruliferous leafhoppers were recollected from the test plants after infection, allowing a sustainable use of the insects. The method thus enables more reliable phenotyping by increasing infection success and testing a greater number of genotypes in a shorter time.

Association of Single Nucleotide Polymorphisms (SNPS) to Black Spot Resistance in Roses

Rose black spot caused by Diplocarpon rosae is the most severe and global disease of garden roses. Breeding of disease resistant varieties is one of the most important goals of modern garden rose breeding. Single nucleotide polymorphisms (SNPs) are the most abundant type of polymorphism found in eukaryotic genomes. SNP markers can be used in a wide variety of applications, including association studies, genetic diversity analysis, and marker-assisted selection in plant breeding programs. The aim of this project was to analyze black spot resistance in a rose association panel through leaf inoculation assays with single isolate pathotypes as well as a field mixture of D. rosae isolates and to establish a relationship between resistance and the available SNP markers. In this study, 96 diverse cultivars of roses were evaluated phenotypically for resistance against black spot through artificial inoculations. 63000 SNPs that were developed in previous studies were used to genetically analyze the cultivars and find associations with resistance against black spot disease using mixed linear model in TASSEL 3.0. Differences in the phenotypic reaction to field mixture of isolates and the Ab13 single conidial isolate of this pathogen were observed between genotypes. One hundred and forty-nine SNPs were found to be significantly associated with resistance against field mixtures of black spot. For the Ab13 single isolate, only one significant SNP was found. These SNPs were mapped on the rose chromosomes, and found on chromosome one, three and four. These associated SNP and Rdr1 markers can be used for marker-assisted selection in breeding for black spot resistance in rose.

First report of Pseudomonas cichorii causing bacterial leaf blight of pocketbook plant (Calceolaria hybrida) in Taiwan.

Pocketbook plants (Calceolaria spp.) are flowering ornamentals often grown as potted plants (Poesch 1937). In December 2022, leaf blight symptoms were observed on 2-mo-old plants of C. hybrida F1 'Dainty'. The disease was found in a nursery in Ren'ai Township, Nantou, and about 20% of the plants exhibited symptoms. Symptomatic plants had brown or gray necrotic lesions of different sizes and shapes, mostly around leaf margins. Lower leaf wilting was also observed (Fig. S1, A and B). Three plants were sampled. Leaf lesions were surface-disinfected with 75% ethanol and cut into smaller pieces in 10 mM MgCl2. After observing bacterial streaming under a microscope, the bacteria were streaked onto nutrient agar (NA). Following 2 days at 28°C, a type of round, creamy white colony predominated on all the plates. Three strains (Calc-A, Calc-B, and Calc-C) were obtained, one from each plant. The strains produced fluorescent pigments on King's B medium and were tested Gram-negative. The strains were characterized with the LOPAT scheme (Schaad et al. 2001). They did not exhibit activities of pectic enzymes, arginine dihydrolase and levan sucrase, but produced oxidase and induced the hypersensitive response in tobacco. DNA was extracted from the strains for PCR amplification of the 16S rDNA with primer pair 27f/1492r as described by Lane (1991). The 16S rDNA sequences were compared with entries in the GenBank database. The sequences obtained (GenBank accession no. OR824302) matched that of Pseudomonas cichorii MAFF 301158 (accession no. AB724288; 1,403/1,403 bp) and were 99% identical to that of DSM 50259T (accession no. CP074349; 1,391/1,405 bp). The strains were also tested with the species-specific primers hrp1a and hrp2a (Cottyn et al. 2011). The amplicons were sequenced and a BLASTn search showed that the sequences (accession no. OR827305) shared the highest identity (99.3%) with that of P. cichorii strain 83-1 (accession no. DQ168848; 848/854 bp) and were 97.3% identical to the sequence of DSM 50259T (accession no. CP074349; 831/854 bp). Calc-A was selected as a representative strain and deposited in the Bioresource Collection and Research Center, Taiwan (reference no. BCRC 81432). Koch's postulates were fulfilled by spray-inoculating a suspension of Calc-A on three 2-mo-old C. hybrida F1 'Dainty' plants. The inoculum was prepared by suspending NA-grown cells in 10 mM MgCl2 including 0.02% Silwet L-77 (OD600 = 0.3; 1.5 x 108 CFU/ml). For the controls, three plants were sprayed with bacteria-free solution. The plants were bagged throughout the experiment and kept in a growth chamber (14/10 h light/dark; 26/24°C day/night). Leaf blight and wilting symptoms developed on all leaves of the inoculated plants after 30 h, but not the controls (Fig. S1, C and D). The pathogen was reisolated from the treatment group, and colony PCR with hrp1a/hrp2a showed that the reisolated strain shared the same sequence with Calc-A to Calc-C. Repeating the inoculation assay produced consistent results. This is the first report of P. cichorii affecting Calceolaria in Taiwan. The bacterium has been reported infecting diverse crops in Taiwan, such as tomato and lettuce (Tsai et al. 2014). Expanding the understanding of the pathogen's potential hosts could help prevent its spread across important crops.

Biocontrol performance of a novel Bacillus velezensis L33a on tomato gray mold and its complete genome sequence analysis

As one of the most destructive diseases, gray mold caused by Botrytis cinerea is harmful to many important economically crops including tomatoes. The use of microorganisms for biological control is considered safe and effective. Here, a newly isolated strain L33a, which showed a 75.22% inhibition ratio against B. cinerea and broad-spectrum resistance to the tested eight phytopathogenic fungi, was characterized by a series of experiments. Strain L33a was identified as Bacillus velezensis based on its biochemical, morphological, and whole-genome profiles. Microstructural analysis revealed that L33a significantly inhibited the mycelial growth of B. cinerea by compromising the integrity of the cell membrane. The acupuncture inoculation assay demonstrated that L33a significantly alleviated disease symptoms caused by gray mold in tomato fruit through downregulation of pathogenicity-related genes of B. cinerea and activation of the transcription of genes involved in the salicylic acid (SA), jasmonic acid (JA), and antioxidant pathways of the tomato fruit. Whole-genome sequencing indicated that the total size of the L33a genome was 4032063 bp, which contained 14 biosynthetic gene clusters reported to be responsible for the biosynthesis of antimicrobial substances. Forty-one compounds were identified in the volatile organic compounds of strain L33a by using Gas Chromatography-Mass Spectrometer (GC-MS) analysis. Among these, 2,3-butanediol, acetoin, 2-tridecanol, and 2-tridecanone were the dominant compounds. Our study revealed that L33a has substantial potential as an effective biocontrol agent, offering valuable insights into postharvest fruit biocontrol.

Inhibitory effect and action mechanism of citral against black rot in pitaya fruit

Blackspot, caused by Alternaria alternata, is a significant postharvest disease in pitaya. Currently, there are limited effective fungicides to manage this disease. This study aimed to evaluate the antifungal activity of citral against the blackspot pathogen and to determine the protective effect of citral on pitaya. Our results demonstrated that citral effectively curtailed the mycelial growth and spore germination of Alternaria alternata, with a minimum inhibitory concentration (MIC) of 0.6 μL/mL. Observations under a light microscope revealed that citral-treated hyphae displayed characteristics such as sparseness, roughness, twisting, shriveling, and irregular internode separation, along with prominent vacuolation. Propidium iodide and 2,7-Dichlorodi-hydrofluorescein diacetate staining results revealed that the mycelia from the MIC citral-treated group emitted robust fluorescence, with the average fluorescence intensities being 4.03 and 8.09 times higher than the control group, respectively. Furthermore, citral treatment resulted in a decline in the lipid content and dry weight of the hyphae, increased nucleic acid and protein leakage, enhanced exosporial conductivity, and augmented superoxide dismutase (SOD) and catalase (CAT) enzyme activities. Additionally, it fostered the accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Fruit damage inoculation assays indicated that an appropriate concentration of citral (1/2 MIC, MIC) could notably diminish the fruit spot diameter expansion. Thus, citral shows promise as a potential alternative fungicide for managing postharvest black rot in pitaya.

Influence of soil moisture conditions on severity of Botryosphaeria branch dieback caused by Lasiodiplodia iraniensis and impact of plant growth in macadamia

Australian macadamias experience outbreaks of Botryosphaeria branch dieback. Extreme environmental conditions are thought to be responsible for the episodic outbreaks of diseases caused by Botryosphaeriaceae species in horticultural ecosystems. However, studies on the interactive effects of biotic and abiotic stresses in macadamia are unknown. Knowledge of management practices that contribute to the severity of the disease would assist in decisions for Botryosphaeria branch dieback control in commercial macadamia orchards. Using an in planta stem wound inoculation assay with Lasiodiplodia iraniensis, we investigated the association of soil moisture conditions as a factor of plant stress with the severity of Botryosphaeriaceae infection in macadamia. Sap flow rate and soil water potential were monitored in 72 macadamia potted cv. HAES 344 plants (18-month-old and 12-month-old grown in pine bark-sand mixture) that were kept under three watering treatments, based on the amount of water used per day (Wd) at the start of the trial, at Wd, ½ of Wd and ¼ of Wd for 14 weeks. A significant differential effect of the watering regimes was observed on disease severity, revealing a strong (R2 > 0.87) negative linear relationship between disease severity and sap flow rates. Necrotic lesions were significantly (P < 0.001) longer in the lowest and highest watering regimes. The average sap flow rate of 0.4 kg h−1 was recorded in the plants under moderately watered conditions, ½Wd regime, whereas 50% and 75% reductions in sap flow rates were recorded at the lowest and highest watering treatments, respectively. Our results indicate that maintaining optimal soil water potential between −50 and −80 kPa through irrigation practices may reduce the risk of Botryosphaeria branch dieback outbreaks in macadamia orchards.