Stem Inoculation Research Articles

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease.

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. & de Not. are the two major forest diseases in the poplar plantations in China, sometimes which can destroy all the poplar seedlings or severely damage mature poplar forests. Hybrid breeding is the most direct and efficient method of controlling and managing tree diseases. However, assessing disease resistance or selecting disease-resistance clones based on In vitro stem inoculation is inefficient, time-consuming, and expensive, limiting the development of hybrid breeding of poplar stem canker disease. In this study, we proposed an alternative method to assess disease resistance to stem canker pathogens through in vivo leaf inoculation. The test materials used in this method can be on 1-year-old poplar saplings or the annual branches of perennial poplars in the greenhouse or the field. The critical step of this alternative method is the selection of inoculating leaves: the 5-7th newly matured leaves might be the most suitable. The second critical step of the leaf inoculation method is to make wounds on plant leaves through needle pierces, providing sufficient lesions to measure disease severity. For the adequate number of leaves produced in the early stage of poplar breeding, this in vivo leaf inoculation contributes to the rapid, accurate, and large-scale screening of the disease-resistance poplar clones to stem canker pathogens. Moreover, this leaf inoculation method will also serve as an efficient method for screening pathotypes of stem canker disease pathogen C. chrysosperma, B.dothidea, or other poplar stem canker pathogens.

Occurrence of Fusarium Crown Rot Caused by Fusarium culmorum on Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximately half of the total sown area of grain crops in XUAR, with 1.14 million hectares in 2021. Fusarium crown rot (FCR) of wheat, caused by Fusarium culmorum (W.G. Smith) Sacc., is one of the most devastating soil-borne diseases known to seriously reduce grain yield (Ma et al. 2024; Saad et al. 2023). In 2016, FCR of wheat, caused by F. culmorum, was firstly identified in Henan Province, China (Li et al. 2016). In June 2023, during the investigation of FCR of wheat in Aksu Prefecture, XUAR, FCR on winter wheat (cv. Xindong 20) was found (82.761349°E, 41.612202°N). The grain-filling period for winter wheat in early June coincided with a period of high temperatures and water demand in Aksu Prefecture. Approximately 8% of the Xindong 20 wheat plants exhibited symptoms of white heads and browning at the stem base, with the disease present in 82% of the wheat fields surveyed. To identify the pathogens, 20 samples of diseased stem basal tissue, each 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, followed by three rinses with sterile water. These samples were then plated onto potato dextrose agar (PDA) medium at 25°C for 5 days. A total of 17 isolates with consistent morphological characteristics were obtained using single-spore technique, with an isolation rate of 85%. The isolated strains exhibited rapid growth on PDA, producing fluffy, pale-yellow hyphae, and accumulating a pale-yellow to dark red pigment on the bottom of the medium. On carnation leaf agar (CLA), these strains formed orange colonies due to the aggregation of a large number of macroconidia. The macroconidia were short and thick, with three to four septa and rounded apical cell, averaging 31.94 to 40.96 × 5.62 to 6.71 μm (Magnification of ×400). Microconidia were not observed. These morphological characters were consistent with those of F. culmorum (Leslie and Summerell. 2006). Two isolates (D-9 and D-11) were selected for molecular identification. The EF-1α gene fragment was amplified using primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') as previously described by O'Donnell et al. (1998). The two 665 bp PCR products were sequenced and submitted to GenBank (GenBank Accession No: PP763247 and PP763248) with 99. 7% identity to the published F. culmorum sequences (e.g., OP985478, OP985477, MG195126, KX702638). The molecular identification was further confirmed by F. culmorum species-specific PCR primers FcOIF/FcOIR (Nicholson et al. 1998). The expected PCR products of 553 bp were produced only in F. culmorum. Strains D-9 and D-11 were used to conduct the pathogenicity experiment on 7-day-old winter wheat (cv. Xindong 20) using drip in the lower stem inoculation method with a 10-μl of 106 macroconidia ml-1 suspension, and the control 7-day-old winter wheat were treated with sterile water (Xu et al. 2017). The experiments were replicated five times in a greenhouse at temperatures ranging from 20℃ to 25℃. After 4 weeks, all inoculated wheat seedlings showed stem base browning or even death. No symptoms were observed on the control plants. The fungus was reisolated from all inoculated wheat plants by the method described above and identified by morphological and PCR amplification using F. culmorum species-specific primers FcOIF/FcOIR. No F. culmorum was isolated from the control wheat plants, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F.culmorum causing FCR on winter wheat in XUAR, China. Considering wheat is the predominant grain crop and plays a pivotal role in grain production in China, necessary measures should be taken to prevent the spread of F. culmorum to other regions.

Multiple Forms of Resistance to the Phomopsis Stem Canker Pathogens Diaporthe helianthi and D. gulyae in Sunflower.

Phomopsis stem canker of cultivated sunflower (Helianthus annuus L.) can be caused by multiple necrotrophic fungi in the genus Diaporthe, with Diaporthe helianthi and D. gulyae being the most common causal agents in the United States. Infection begins at the leaf margins and proceeds primarily through the vasculature, progressing from the leaf through the petiole to the stem, resulting in formation of brown stem lesions centered around the petiole. Sunflower resistance to Phomopsis stem canker is quantitative and genetically complex. Due to the intricate disease process, resistance is possible at different stages of infection, and multiple forms of defense may contribute to the overall level of quantitative resistance. In this study, sunflower lines exhibiting field resistance to Phomopsis stem canker were evaluated for stem and leaf resistance to multiple isolates of D. helianthi and D. gulyae in greenhouse experiments, and responses to the two species were compared. Additionally, selected resistant and susceptible lines were evaluated for petiole transmission resistance to D. helianthi. Lines with distinct forms of resistance were identified, and results indicated that responses to stem inoculation were strongly correlated (Spearman's coefficient 0.598, P < 0.001) for the two fungal species, while leaf responses were not (Spearman's coefficient 0.396, P = 0.076). These results provide a basis for genetic dissection of distinct forms of sunflower resistance to Phomopsis stem canker and will facilitate combining different forms of resistance to potentially achieve durable control of this disease in sunflower hybrids.

First report of Cassava Bacterial Blight caused by Xanthomonas phaseoli pv. manihotis in the Amazonian forest of Ecuador.

Xanthomonas phaseoli pv. manihotis (Xpm) is a plant pathogenic bacterium known as the causal agent of cassava bacterial blight (CBB). CBB is the most limiting bacterial disease affecting cassava (Manihot esculenta Crantz), characterized by diverse symptoms including angular water-soaked leaf lesions, blight, wilting, stem exudates, stem cankers and dieback. CBB has been reported in most cassava-growing regions around the world, and, under conducive conditions, crop yield losses can reach up to 100% (Zárate-Chaves et al. 2021). While Xpm genetic diversity is remarkably high in South America (Bart et al. 2012) and cassava originates and was domesticated in the Amazon basin (Allem 2002), reports of CBB in the Amazonian region are missing. To fill this gap, in October 2018 we surveyed for CBB symptoms in cassava fields of the Orellana Province, located in the Amazon forest of the Republic of Ecuador. Adult cassava plants exhibiting typical angular, water-soaked leaf lesions were found in polyculture plots, i.e. intercrops of cassava with other species such as plantains and fruit trees (a.k.a. chakras). After surface disinfection with 5% sodium hypochlorite followed by 70% ethanol, white Xpm-like colonies were isolated from diseased leaf tissues of four plants on YPGA medium (yeast extract, 5 g/l; peptone, 5 g/l; glucose, 5 g/l; agar-agar, 15 g/l) supplemented with cephalexin (40 mg/l) and cycloheximide (50 mg/l). Pathogenicity tests were performed on peat-potted, 2-month-old cassava plants of the cultivar 60444. Bacterial suspensions were adjusted to an OD600 of 0.2 (2 × 108 CFU/ml) in sterile 10-mM MgCl2 and syringe infiltrated in fully-expanded leaves. In parallel, 20 µl of each bacterial suspension adjusted to an OD600 of 0.02 (2 × 107 CFU/ml) were inoculated on stems inside a hole previously punched with a sterile needle in the junction of the third-top petiole. Sterile 10-mM MgCl2 was used for mock inoculations in both leaves and stems, and experiments were replicated in three plants. Plants were incubated in a greenhouse at 28 ± 1°C with a 12-h photoperiod. Infiltrated leaves developed watersoaking 3 days post inoculation, while wilted leaves, stem exudates, and dieback were observed 21 days after stem inoculation. Control plants remained symptomless. White Xpm-like colonies were re-isolated from symptomatic leaves (Fig S1). One colony of each of the four Xpm isolates (before and after re-isolation) was assessed using diagnostic PCRs (Bernal-Galeano et al. 2018; Flores et al. 2019), using strain Xam668 as positive control. All four candidates were positive for both diagnostic tools. The sequences of the housekeeping genes atpD, dnaK, efp, glnA, gyrB and rpoD of our isolates were extracted from full genome sequences obtained through Oxford Nanopore Technologies (ONT) (GenBank OR288194 to OR288217) and compared to their homologs in four close Xanthomonas species and a reference Xpm strain (Table S1). The sequences of the tested strains aligned with that of Xpm CIO151 (GCA_004025275.1) (Arrieta-Ortiz et al. 2013) with nucleotide identity above 99.92% (Fig S2). The four strains were named CIX4169, CIX4170, CIX4171 and CIX4172, stored in the IRD Collection of Xanthomonas, where they are available upon request. To our knowledge, this is the first report of CBB in the Amazonian region and in Ecuador, where cassava is a central element for local culture and economy. Further surveys will be necessary to evaluate the distribution and prevalence of CBB in other ecozones of Ecuador where cassava is cultivated.

Phenotypic Evaluation of a Lemon Hybrid Population to Identify Sources of Resistance to Plenodomus tracheiphilus

Mal secco, caused by the fungus Plenodomus tracheiphilus, is a xylem disease that is a limiting factor for lemon production in the Mediterranean. Resistance or field tolerance are major goals for lemon breeders; however, there is scant information regarding the heritability of mal secco resistance in breeding populations. As with other vascular diseases, phenotyping is the bottleneck for ascertaining resistance and susceptibility, and a validated protocol for greenhouse phenotyping would be valuable to accelerate the selection of tolerant trees before field evaluation. We report phenotyping of 148 hybrids of Khasi papeda (Citrus latipes; tolerant to mal secco) × lemon (susceptible to the disease) in field and greenhouse conditions. Field evaluation was performed on all hybrids for 2 to 3 consecutive years on trees subjected to high natural-pathogen pressure. Detection of the fungal infection was performed by visual observation and real-time polymerase chain reaction (PCR). The first infections occurred ≈6 months after planting, but 2 years of observations were needed for a reliable estimation of susceptibility. The spread of the disease did not occur uniformly throughout the plot, with patterns of spread within rows, probably resulting from infections from plant to plant. The possible errors in the estimation of susceptibility as a result of the uneven distribution of infections in the plot were reduced by using more than one replicate tree per hybrid. The correlation between phenotyping scores and cycle threshold values was weak (r = –0.48, P &lt; 0.001). Three years after planting, hybrids clustered into three groups—susceptible, tolerant, and intermediate—based on symptom progression. A subset of 65 self-rooted hybrids was also subjected to stem inoculation in an unheated greenhouse, with two to seven biological replicates per hybrid. Three months after inoculation, the samples were monitored for symptoms appearance and subjected to real-time PCR pathogen quantification. We observed a weak (r = 0.41) but significant (P &lt; 0.001) correlation between phenotypes in the field and the greenhouse, indicating that, in our conditions, field evaluation remains the best method for phenotyping. However, artificial inoculations might help to discard the highly susceptible hybrids before field evaluation.

Distribution of Charcoal Rot of Soybean, its Influencing Factors and Pathogenic Variabilities in Different Regions of Madhya Pradesh

Background: Macrophomina phaseolina (Tassi) Goid is a necrotrophic soil-inhabiting fungus that causes disease in several economic plants worldwide and has now become a serious threat to soybean cultivation in central India. Methods: The random surveys were done during 2018 and 2019 to determine the present status of charcoal rot (M. phaseolina) in key soybean growing districts of Madhya Pradesh. Sixteen isolates of pathogen were characterized based on morpho-cultural and pathogenic variability by using cut stem inoculation techniques at J.N.K.V.V., Jabalpur. Result: Charcoal rot was prevalent in all surveyed sixteen districts of Madhya Pradesh. Out of six, Kymore plateau and Satpura hills (44.5%), Satpura plateau (42.5%), Central Narmada Valley (42.0%) and Vindhyan plateau agroclimatic zones (35.0%) were identified as favouable zones for charcoal rot. Malwa plateau (16.25%) and Northern hill region (24.5%) had low incidences of charcoal rot. Among varieties, JS 95-60, JS 93-05 and JS 20-29 were highly affected, whereas, JS 20-34, JS 20-98 and JS 20-69 were least affected. Incidence of charcoal rot was comparatively higher in the fields that had Maize-Chickpea (26.32%), Maize-Wheat, Soybean- Pea and Soybean - Chickpea cropping patterns in the previous year and lowest in Rice-Chickpea (12.00%) and Rice - Wheat (12.45%) cropping patterns. The incidence of charcoal rot was partially higher in the fields with light soil (21.3%) than in heavy soil (19.0%). Isolates investigation revealed that isolates from Jabalpur, Hoshangabad, Chhindwara and Sagar were fast-growing variables and highly aggressive in developing necrotic lesions on the cut stem of soybean. This investigation could be instrumental in forewarning sensitive areas of charcoal rot, varietal options and crop rotation to be followed to minimize the incidence of charcoal rot. Moreover, identified variable aggressive isolates could be utilized in genotype resistance screening programs.

New fungicides for managing Phytophthora diseases of tree crops with foliar and soil applications

Four new modes of action, oxathiapiprolin, fluopicolide, mandipropamid, and ethaboxam, have or are pending registrations on multiple tree crops in the USA including avocado, citrus, almond (and other tree nuts), and stone fruits to be used in sustainable anti-resistance rotation and mixture programs for managing Phytophthora diseases. All four fungicides are highly toxic in vitro against species of Phytophthora from different tree crops. Efficacy of foliar applications of oxathiapiprolin and mandipropamid for the management of citrus brown rot persisted for over 10 weeks. Still, residues could be removed to non-detectable levels by postharvest washing and brushing of fresh-market fruit. The four fungicides were highly effective in reducing root rot and crown/trunk cankers on tree crops and demonstrated acropetal systemic movement through the xylem after soil application and absorption by almond, citrus, and cherry roots. This was substantiated by reduced lesion size in stem inoculations of potted trees that were treated by soil applications or by in vitro bioassays and HPLC–MS–MS analyses of root, stem, and leaf extracts. Using an adapted Bromilow model that is based on the partition coefficient logP that indicates the lipo- or hydrophilicity and the acid dissociation constant pKa, mobility of ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin in plants was predicted, and this was supported by our studies. Mandipropamid and a mandipropamid–oxathiapiprolin pre-mixture are now registered as foliar applications for managing citrus brown rot, whereas oxathiapiprolin and fluopicolide are registered, and ethaboxam is pending registration as soil applications to control citrus root rot. Oxathiapiprolin has also been registered for soil use on almond and other nut crops, as well as avocado, and soil applications of mandipropamid are planned to be restricted to nursery use of multiple crops.

Evaluation of F2 Mapping Population for Stem Rot Tolerance in Tossa Jute (Corchorus olitorius)

Present investigation was undertaken at ICAR-CRIJAF, Barrackpore, West Bengal, India during summer season (Apr–July) of 2020 to identify the jute breeding lines resistant to stem rot which is one of the major problems in jute production. The identified resistant and susceptible jute accessions (RS-6 and OIJ-272, respectively) were crossed to develop a mapping population for the disease. The resultant F2 population comprising 125 plants was screened for resistance to stem rot disease caused by Macrophomina phaseolina following artificial inoculation. The progress of infection was measured as the length of the infected stem portion (i.e., lesion length in cm) after 7, 14 and 21 days of inoculation (time replication). After stem inoculation investigation indicated that in the F2 population of the cross OIJ-272×RS-6, only 14 plants (PL-100, PL-40, PL-99, PL-33, PL-89, PL-119, PL-1, PL-43, PL-118, PL-121, PL-6, PL-11, PL-55 and PL-86) were found moderately resistant (lesion length 2.0–3.0 cm). In contrast, 10 plants (PL-4, PL-31, PL-19, PL-27, PL-3, PL-50, PL-18, PL-26, PL-47, PL-51) were found highly susceptible (lesion length &gt;5.0 cm). The remaining 101 plants exhibited moderate susceptible symptoms. While the parent OIJ-272 exhibited high susceptibility to stem rot with an average lesion length of 5.53 cm, the parent RS-6 exhibited moderate resistant reaction with an average lesion length of 2.30 cm. Because of good resistance under artificial stem inoculation of this fungi, these 14 F2 plants of the cross can be further exploited for a resistance breeding programme against this deadly disease.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 μm x 3.77-6.84 μm (average 22.90 μm x 5.20 μm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.

The Fungal Pathogen Gnomoniopsis castaneae Induces Damaging Cankers in Multiple Domestic Fagaceae Species.

Gnomoniopsis castaneae is internationally recognized as a destructive pathogen of chestnut species. Primarily associated with nut rot, it has also been associated with branch and stem cankers of chestnut and as an endophyte of multiple additional hardwood species. The present study evaluated implications of the recently reported United States presence of the pathogen for domestic Fagaceae species. Stem inoculation assays of Castanea dentata, C. mollissima, C. dentata × C. mollissima, and Quercus rubra (red oak) seedlings were utilized to examine the cankering ability of a regional isolate of the pathogen. The pathogen induced damaging cankers in all assessed species and significant stem girdling in all chestnut species. No previous study has associated the pathogen with damaging infection in Quercus species, and its presence in the United States has the potential to compound ongoing chestnut recovery programs and oak regeneration problems within forest systems.

First Report of Melon Soft Rot Disease Caused by Pectobacterium brasiliense in Korea

In May 2021, characteristic soft rot symptoms, including soft, watery, slimy, black rot, wilting, and leaf collapse, were observed on melon plants (&lt;i&gt;Cucumis melo&lt;/i&gt;) in Gokseong, Jeollanam-do, Korea. A bacterial strain, designated KNUB-06-21, was isolated from infected plant samples, taxonomically classified, and phylogenetically analyzed using 16S rRNA and housekeeping gene sequencing. Strain KNUB-06-21 was also examined for compound utilization using the API ID 32 GN system and strain KNUB-06-21 was identified as Pectobacterium brasiliense. Subsequent melon stem inoculation studies using strain KNUB-06-21 showed soft rot symptoms similar to field plants. Re-isolated strains shared phenotypic and molecular characteristics with the original &lt;i&gt;P. brasiliense&lt;/i&gt; KNUB-06-21 strain. To our knowledge, ours is the first report of &lt;i&gt;P. brasiliense&lt;/i&gt; causing melon soft rot disease in Korea.

Using Transferable Eucalypt Microsatellite Markers to Identify QTL for Resistance to Ceratocystis Wilt Disease in Eucalyptus pellita F. Muel. (Myrtales, Myrtaceae)

The deployment of Eucalyptus pellita trees that are resistant to Ceratocystis manginecans is essential for the commercial plantations and therefore the sustainability of forest industries in Southeast Asia that utilize this resource. Current screening procedures are time-consuming and expensive but could be expedited with the aid of marker-assisted selection and breeding. The identification of genotypes with resistance to the disease may be facilitated if microsatellite markers developed in other Eucalyptus species are transferable and can be linked to quantitative trait loci (QTL) for disease resistance. This possibility was tested in 111 full-sib progenies and their parents by genotyping with 49 microsatellite markers developed in other Eucalyptus species. Disease development was assessed after stem inoculation with C. manginecans isolate Am60C. The disease index (DI) varied from 0 to 20% of stem length. There was a continuous distribution of resistant and susceptible seedlings with 60% in the resistant category. Of the 30 acceptable markers, 17 (56%) defined two linkage groups (LG). In each LG, one QTL with a significant logarithm of odds (LODs &gt; 13) was identified. The transferability of microsatellite markers developed in other Eucalyptus species facilitated the rapid identification of LGs and QTLs in E. pellita. To further refine the linkage map, the testing of more microsatellite markers and a larger population of progenies are required.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Sławiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

Determination of Phytophthora Species Causing Root and Crown Rot on Tomatoes Grown in Antalya Province and Reactions of Some Tomato Genotypes against Phytophthora nicotianae

Antalya province is the main center of vegetable production in Türkiye. Tomato comes first in terms of crops cultivated under greenhouse. Phytophthora species causing root and crown rot are among the factors negatively affecting tomato yield and quality. This research aimed to determine the prevalence of root and crown rot of tomatoes grown in Antalya province and to identify Phytophthora species causing disease. During surveys performed in 170 tomato greenhouses, plant and soil samples were taken from the areas where root and crown rot, stem blight, and drying symptoms were observed. Disease prevalence and incidence in the investigated greenhouses were 25.88% and 4.87%, respectively. Phytophthora symptoms were not observed in the greenhouses in Demre and Kepez districts, while the highest disease prevalence was found in Elmalı district with 75%. Eighty of 84 Phytophthora isolates were identified as P. nicotianae and four as P. capsici, according to their cultural, morphological and molecular characterisation. Virulence of 18 selected isolates were determined by using stem inoculation technique and all isolates caused lesions with different lengths on tomato stems. The reactions of 22 tomato genotypes in the gene pool of BATEM against P. nicotianae were also investigated and the genotype DT-15 was found as the most susceptible genotype with the largest lesions, while A-286 was the most resistant genotype. This study formed the basis for further studies on tomato breeding and integrated disease management.

First Report of Botryosphaeria dothidea Causing Stem Blight of Dioscorea oppositifolia in China.

Dioscorea oppositifolia Thunb. (Chinese yam) is one of the traditional foods and medicinal plants in China. It has nutritional and medicinal value and plays an important role in treatment of diabetes and hypertension. In 2018, stem blight was first observed on the stalks of D. oppositifolia in fields of Anguo City (115°27' N; 38°46' E), Hebei Province, China. Over 400 plants were surveyed in four fields, and nearly 30% of the plants were infected. At the initial stage of the disease, there were dark brown spots on the stems and in later stages the leaves and stems withered. To identify the pathogen, 10 symptomatic stalks were collected, and one diseased area was taken out from each sample. Small square stalk pieces (3 to 5 mm) were obtained with sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min, followed by washing in sterile water three times, then pieces were transferred to potato dextrose agar (PDA) plates for 7 days at 25°C. The fungal isolates were purified by single-spore isolation. A total of three species of fungi were isolated, and initial pathogenicity tests found that one fungal species could cause the disease symptoms on D. oppositifolia stems. This pathogen was grown on PDA plates in the dark at 25 °C for 10 days. In the beginning, the colonies were white, and as the culture time was extended, the color of the colonies became darker and then became black. Conidia forming on pycnidia were one-celled, hyaline, aseptate, and ovoid, with dimensions of 4.6 to 7.6 × 2.6 to 4.8 μm (n=100). Mycelial DNA was extracted from a 7-day-old culture, and PCR amplifications were performed using primers ITS1/ITS4 and β-tubF/β-tubR (Glass and Donaldson 1995; White et al. 1990). BLAST searches at GenBank showed 100.00% nucleotide sequence identity for the ITS sequence with Botryosphaeria dothidea strain sdxf6 (MG282093; 545/545 bp) and for β-tubulin 99.76% identity with B. dothidea strain SD-B8 (KP183131; 411/412 bp). Sequences from these regions were deposited in GenBank (ITS: OP104323; β-tubulin: OK669147). Morphological and molecular results confirmed this species as B. dothidea (Angelica et al. 2017; Bernard et al. 2004). To inoculate plants, pathogen was grown on PDA at 25°C in the dark for 15 days, after which a spore suspension (3×105 spores/mL) was prepared by flooding the agar surface with sterilized double-distilled water. Pathogenicity tests were conducted by stem inoculation of 6-month-old healthy D. oppositifolia plants. The stems were wounded by lightly rubbing with a steel sponge, and the wounded stem was wrapped in sterile cotton treated with 1 mL of the spore suspension, then the plants were covered with plastic to maintain a moist environment for 72 h. Control plants were inoculated with sterile water. Inoculated and control plants (ten each) were kept in a moist chamber (25°C, 16-h light and 8-h dark period, 75% relative humidity). After 15 days, all of the inoculated plants showed dark brown spots on the stems, and the symptoms were the same as those in the field, while the controls were healthy. After 30 days, all of the inoculated but none of the control D. oppositifolia plants showed leaf wilting or leaf withering. Isolates from the inoculated and infected leaves were identified as B. dothidea by DNA sequencing with primers ITS1/ITS4 and β-tubF/β-tubR, fulfilling Koch's postulates. To our knowledge, this is the first report of B. dothidea causing stem blight on D. oppositifolia. The disease poses a threat to the production of D. oppositifolia, and management strategies need to be developed.

Biocontrol Potential of an Endophytic Pseudomonas poae Strain against the Grapevine Trunk Disease Pathogen Neofusicoccum luteum and Its Mechanism of Action.

Grapevine trunk diseases (GTDs) impact the sustainability of vineyards worldwide and management options are currently limited. Biological control agents (BCAs) may offer a viable alternative for disease control. With an aim to develop an effective biocontrol strategy against the GTD pathogen Neofusicoccum luteum, this study investigated the following: (1) the efficacy of the strains in suppressing the BD pathogen N. luteum in detached canes and potted vines; (2) the ability of a strain of Pseudomonas poae (BCA17) to colonize and persist within grapevine tissues; and (3) the mode of action of BCA17 to antagonize N. luteum. Co-inoculations of the antagonistic bacterial strains with N. luteum revealed that one strain of P. poae (BCA17) suppressed infection by 100% and 80% in detached canes and potted vines, respectively. Stem inoculations of a laboratory-generated rifampicin-resistant strain of BCA17 in potted vines (cv. Shiraz) indicated the bacterial strain could colonize and persist in the grapevine tissues, potentially providing some protection against GTDs for up to 6 months. The bioactive diffusible compounds secreted by BCA17 significantly reduced the spore germination and fungal biomass of N. luteum and the other representative GTD pathogens. Complementary analysis via MALDI-TOF revealed the presence of an unknown cyclic lipopeptide in the bioactive diffusible compounds, which was absent in a non-antagonistic strain of P. poae (JMN13), suggesting this novel lipopeptide may be responsible for the biocontrol activity of the BCA17. Our study provided evidence that P. poae BCA17 is a potential BCA to combat N. luteum, with a potential novel mode of action.

A modified and effective stem inoculation technique for artificial screening against Sclerotinia sclerotiorum in mustard

AbstractSclerotinia stem rot (SR) disease emerged as a potential threat in most mustard‐growing regions globally. Sclerotinia sclerotiorum, a necrotrophic pathogen, causes considerable damage to the crop under congenial environments. The sclerotia even under adverse conditions can survive up to 5 years in the soil. The use of resistant sources is the only mean to combat the pathogen. The development of an effective inoculation technique can facilitate the identification of new sources of resistance rapidly. Investigations on resistant sources using Potato Dextrose Agar (PDA) medium for artificial stem inoculation are ongoing under the All India Coordinated Research Project (AICRP) on rapeseed‐mustard for many years but no resistant sources could been identified. In the present study, two non‐injury stem inoculation techniques using sorghum grains and PDA medium as a basal medium were tested on 11 brassica entries for up to three consecutive years. Results revealed that sorghum grain‐based inoculum was found efficient for disease development and lesion formation in brassica entries compared with PDA‐based inoculum. Among the entries, a maximum disease incidence of 42.3% was recorded in entry DRMR 5206 followed by DRMRSJ‐20, DRMRSJ‐22 and DRMR 2017‐8 with 42.1%, 41.8% and 41.3% SR incidence, respectively. In PDA disc inoculation maximum SR incidence of 30.8% was observed in DRMRSJ‐26 followed by DRMR 5206, DRMRSJ‐25 and DRMRSJ‐2017‐18 with 27.5%–24.9%, respectively. Lesion length was more in entries inoculated with fungal mycelium multiplied on sorghum grains compared with the PDA disc inoculation method. The statistical analysis and results of p‐value (&lt;.00001) showed a significant difference between tested methodologies. Thus, the sorghum grain‐based inoculation technique is an appropriate, economic and effective non‐injury inoculation technique for artificial screening against S. sclerotiorum in the mustard crop. This technique can be used to screen a large number of brassica entries more efficiently with less labour and time.

First report of Diaporthe gardeniae causing branch blight of Gardenia jasminoides Ellis in Zhejiang Province, China.

Gardenia jasminoides Ellis, an important Chinese medicine, is cultivated on approximately 1,400 hectares in China. From August to October 2016, a severe disease affecting leaves, stems, and fruits of G. jasminoides, occurred in Cangnan (120°39'E, 27°48'N), Zhejiang province. Infected leaves or stems became shriveled, and in severe cases, young fruits presented red-brown or yellow necrotic lesions with numerous black spots. More than thirty diseased fruit and stem samples were collected, ten diseased fruits were surfacedisinfected (70% ethanol for 30 s, 1.5% sodium hypochlorite for 1 min) and kept at about 25℃ for 24 h with 80% humidity. The conidial suspension (105 conidia/ml) made of creamy drops secreted from the lesion black spots was spread onto PDA (Potato-Dextrose-Agar) and incubated at 25℃ in the dark for 7 d. Only one isolate (step01) was suspected to be the target pathogen, and other three isolates were Alternaria sp. The colony of step01 was white to grayish with an irregular edge on the front and a white to brown spiral grain on the back. Black pycnidia, produced after 20 d, were globose to subglobose, individual or overlapped, with an ostiole secreting a creamy conidial suspension. Alpha-conidia were aseptate, hyaline, oval to oblong with two oil balls, 7.4-15.9×2.4-4.5 µm (average 10.2×3.3 µm); beta-conidia were hyaline, aseptate, linetype, straight or slightly curled, 15.3-26.5×1.3-2.5 µm (average 20.8×1.6 µm). This isolate resembled Diaporthe sp. (Hansen and Barrett 1938). For species identification, DNA was extracted (Sangon Biotech Rapid Fungi Genomic DNA Isolation Kit - B518229)，and the internal transcribed spacer region (ITS), elongation factor (EF1-α), β-Tubulin (TUB), and histone H3 (HIS) of step01 were amplified using the primer pairs ITS1/ITS4, EF1-728F/ EF1-986R, BT-2a /BT-2b and CYLH3F/CYLH3R, respectively (Udayanga et al 2014, Huang et al. 2015).These sequences were submitted to GenBank as KY797655 (ITS), MF158048 (EF1- α), MF158049 (TUB), and MF158050 (HIS). In comparison with the other sequence of Diaporthe sp. using MEGA7.0 (maximum likelihood, bootstrap replications=1,000), step01 showed 100% identity with D. gardeniae. Based on their morphology and molecular identification, step01 was identified as D. gardeniae (syn. Phomopsis gardeniae). Pathogenicity was tested on three one-year-old G. jasminoides plants by stem inoculation. Two or three stems per plant were inoculated by binding a mycelial plug (5 ×12 mm), covered by humid cotton and plastic film, to the tender stem. A total of two plants were treated. Plants were kept at about 25℃ for 4 weeks. Control plants were inoculated with PDA plugs. Leaf blight started from the apex, extended to the stalk, and the leaves finally fell off. Three months after inoculation, symptoms developed on the underlying leaves, the stem was withered with black spots, a pattern like that observed in the field. No symptoms appeared in the control leaves. Five identical colonies were re-isolated from symptomatic tissues and identified again as D. gardeniae, fulfilling the Koch's postulates. Several fungi are associated with canker, leaf spot, and fruit rot in Gardenia throughout China, including Pestalotiopsis sp. (Huang et al. 2006), Botryosphaeria dothidea (Dong et al. 2016), and Phoma sp. (Luo et al. 2016). To the best of our knowledge, this is the first report of D. gardenia infecting G. jasminoides Ellis in Zhejiang Province, China.

Phytophthora pseudocryptogea, P. nicotianae and P. multivora Associated to Cycas revoluta: First Report Worldwide.

A dieback was observed on three-year-old pot-grown plants of Cycas revoluta in Sicily (Italy). Symptoms, including stunting, yellowing and blight of the leaf crown, root rot and internal browning and decay of the basal stem, closely resembled the Phytophthora root and crown rot syndrome, common in other ornamentals. Isolations from rotten stem and roots, using a selective medium, and from rhizosphere soil of symptomatic plants, using leaf baiting, yielded three Phytophthora species, P. multivora, P. nicotianae and P. pseudocryptogea, were obtained. Isolates were identified by both morphological characters and DNA barcoding analysis, using three gene regions: ITS, β-tub and COI. Phytophthora pseudocryptogea was the sole species isolated directly from the stem and roots. The pathogenicity of the isolates of the three Phytophthora species was tested on one-year-old potted plants of C. revoluta, using both stem inoculation by wounding, and root inoculation through infested soil. Phytophthora pseudocryptogea was the most virulent and, like P. nicotianae, reproduced all the symptoms of natural infections, while P. multivora was the least virulent and induced solely very mild symptoms. Phytophthora pseudocryptogea was identified as the causal agent of the decline of C. revoluta, as it was re-isolated from both the roots and stems of artificially infected symptomatic plants, thus fulfilling Koch's postulates.

Development of Screening Assays for Pathogen Virulence and Resistance to Phytophthora Root Rot and Wilting Complex in Raspberry.

Phytophthora root rot and wilting complex (PRRW) of red raspberry, caused primarily by Phytophthora rubi, is an economically important disease in British Columbia (BC) and in raspberry producing regions globally. Reliable, rapid, and efficient screening methods are lacking for evaluating germplasm for potential disease resistance in raspberry breeding programs as well as for screening pathogen isolates for virulence. The objective of this study was to compare various screening methods for efficiency and rapidity in inducing symptoms of disease to identify the most suitable approach. We compared several intact plant root inoculation (IPRI) assays, detached stem assays, and an intact plant stem inoculation (IPSI) assay. A virulent isolate of P. rubi was inoculated in two commercial cultivars: 'Chemainus' (susceptible to PRRW) and 'Cascade Bounty' (moderately resistant to PRRW). For IPRI assays, days to first symptom development, plant wilt progression, and root assessment were recorded. For detached stem tissue and IPSI assays, days to first visible lesions and lesion size were assessed. Experiments were arranged in a completely randomized design with three replications in each experiment. Three IPRI assays produced reliable symptoms in both cultivars. Among the detached stem assays, a node inoculation method performed better than other methods. Detached stem assays are useful for rapid pathogenicity testing of P. rubi, whereas IPRI assays are better for screening germplasm for disease resistance. Overall, this study identified several assays that can be used for conducting studies on pathogen phenotypic diversity (pathogenicity and virulence tests) and screening raspberry cultivars, germplasm, and breeding materials for response to PRRW.