Stem Wilt Research Articles

Detection and Characterization of Lasiodiplodia pseudotheobromae Associated with Stem Wilt on Ficus hirta (Vahl) and Its Fungicidal Sensitivity

Ficus hirta Vahl is an important medicinal and edible plant in southern China. Typical green wilting on leaves and brown necrotic spots on the stems were observed since mid-June 2022 in an F. hirta plantation in Danzhou, Hainan (China). The disease rapidly developed, causing stem withering and plant death. The disease incidence varied from 45 to 85% prevalence, and the average disease index was 47 in the period of outbreak during June to December. Relevant hypothetical fungi were isolated from naturally infected wilt tissues, and their pathogens were preliminarily confirmed to be Lasiodiplodia pseudotheobromae through hypothetical fungal culturing, morphological characteristic observations, and pathogenicity testing on F. hirta plants. The phylogenetic tree constructed based on partial ITS, translation elongation factor (TEF1-α), and the β-tubulin gene (TUB2) further confirmed the identity of the pathogen as L. pseudotheobromae. Further research on the biological characteristics of L. pseudotheobromae showed that the optimal temperature for the growth of L. pseudotheobromae was PDA medium, with a temperature of 30 °C and pH of 6. Peptone and fructose were the optimal nitrogen and carbon sources for it. In vitro efficacy testing showed that among eleven fungicides, fluazinam and prochloraz had the highest mycelial growth inhibition, with an EC50 of 0.0477 µg/mL and 0.0996 µg/mL, respectively. And the two fungicides showed significant control on the stem wilt of F. hirta in a pot. To our knowledge, this is the first comprehensive report on the pathogen identification and biological characteristics of L. pseudotheobromae infecting the stem wilt of F. hirta in China. Our results provide important information for developing effective management measures and controlling this disease.

Cucumber Disease Image Classification with A Model Combining LBP and VGG-16 Features

Cucumber (Cucumis sativus) is a significant horticultural crop worldwide, highly valued for both fresh consumption and processing. However, cucumber cultivation faces challenges due to diseases that can substantially reduce yield and quality. Diseases like leaf spots, stem wilt, and fruit rot are caused by pathogens including viruses, bacteria, and fungi. Traditionally, disease detection in cucumbers is performed manually, which is time-consuming and inefficient. Therefore, developing machine vision-based models using Deep Learning (DL) and Machine Learning (ML) for early disease detection through image analysis is crucial for assisting farmers. While many studies on plant disease classification using various DL and ML models show optimal results, research on cucumbers has mostly focused on leaf diseases. This study aims to optimize cucumber disease image classification by developing a model that combines Local Binary Pattern (LBP) texture features and VGG-16 convolutional features. The dataset used, Cucumber Disease Recognition Dataset consists of 8 classes of cucumber plant disease images covering leaves, stems, and fruits. This study classifies cucumber plant disease images using Random Forest (RF) combined with LBP texture features and VGG-16 visual features and compares its performance with models using VGG-16, LBP+RF, and VGG-16+RF on the same dataset. The results show that the proposed model achieved a precision of 84.7%, recall of 84%, F1-Score of 83.8%, and accuracy of 84%. These results outperform the comparative models, demonstrating the effectiveness of the combined approach in classifying cucumber plant diseases.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Euphorbia lathyris in China.

Euphorbia lathyris L. is a biennial herb in the Euphorbiaceae that has been used as a medicinal plant. It is distributed or cultivated worldwide, and the seeds of E. lathyris are the main source of ingenol, which is the precursor of Picato, the first medicine approved by USFDA for the treatment of solar keratosis (Abramovits et al. 2013). However, the production of E. lathyris can be severely hampered by the occurrence of plant diseases. Between 2020-2022 (specifically in October-November of each year), anthracnose-like symptoms were observed on E. lathyris in fields (E 118°49'50″, N 32°3'33″) in Nanjing, Jiangsu Province, China. The incidence of E. lathyris with disease symptoms was between 25%-30% (n = 100). The lesions on the leaves were evident initially as dark brown spots, which expanded into larger necrotic spots, finally resulting in leaves withering and dropping off. In severe cases, stem wilting was also observed. To determine the causal agent, we collected diseased leaf samples (n = 20) from different E. lathyris plants in the field (~ 1800 m2). After cleaning, the junctions of the diseased and healthy parts were excised and sterilized in 75% ethanol for 20-25 seconds, and rinsed with sterile water. After that, they were transferred onto potato sucrose agar (PSA) plates and placed at 25℃ for 3-4 days, until fungal growth was evident. The fungus was purified by recovering single conidia and growing them on PSA (Hu et al. 2015). A consistent fungal colony, based on morphological characteristics, was recovered from 17 samples. The colony color was initially white, green in the middle, and gradually changed into gray green as the colony matured. Conidia were transparent and cylindrical (22-28 µm × 7-9 µm, n = 50). Five loci informative (ITS, TUB, ACT, GAPDH, and CHS-1) (Weir et al. 2012) for Colletotrichum spp. identification were sequenced from two isolates ELC-1 and ELC-2 obtained from different plant individuals. Compared with a reference isolate (Colletotrichum gloeosporioides ZH3), the GAPDH, CHS-1, and TUB2 sequences of ELC-1 and ELC-2 showed 95% (263 bp out of 275 bp), 98% (295 bp out of 299 bp), and 99% (711 bp out of 712 bp and 717 bp out of 719 bp) similarity, respectively. The ITS sequence identities were 100% (577 bp out of 577 bp) and 99% (594 bp out of 597 bp), while the ACT sequence identities were 100% (281 bp out of 281 bp) and 98% (279 bp out of 284 bp). All sequences have been deposited in Genbank database (OR865865-OR865866 and OR873625-OR873632). After performing phylogenetic analysis with Mega 11, the pathogen was confirmed as C. gloeosporioides. To fulfil Koch's postulates, we sprayed six-week-old healthy plants with a conidia suspension of C. gloeosporioides (106 spores/mL) or sterile water (serve as control). The inoculated plants were placed at 25℃, 100% relative humidity, and 12-h photoperiod (Zhang et al. 2021). Six plants were inoculated for each treatment, and the experiment was repeated three times. After 6-8 days, the plants inoculated with C. gloeosporioides showed similar symptoms to those observed on diseased plants in the field, while the control plants remained healthy and free of disease. The pathogens were then re-isolated and identified as C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on E. lathyris. Anthracnose may cause significant yield losses in E. lathyris production, and our results will provide experimental and theoretical basis for the management of the disease.

First Report of Fusarium Wilt on lily (Lilium 'Tresor') Caused by Fusarium armeniacum in Jiangsu Province, China.

In June 2021, a disease of stem and leaf rot was observed on lily cultivar 'Tresor' with approximately 20% disease incidence in fields at Huaiyin District (119°04'N, 33°63'E) of Huaian County, Jiangsu Province. The roots and bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants (Fig. 1A and Fig. 3C). To isolate the pathogen, necrotized plant tissues were surface sterilized with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s and rinsed with sterile water. About 4 mm × 4 mm of diseased tissues were placed on potato dextrose agar (PDA) followed by incubation at 25°C in the dark for 5 days. The pure cultures were obtained by the hyphal-tip method. A total of four fungal isolates with similar colony characteristics were recovered. To determine the identity of the four isolated fungal isolates, genomic DNA was extracted using the method previously described (Khan et al. 2021), the sequences of the internal transcribed spacer (ITS), the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were analyzed with primers ITS1/ITS4 (White et al. 1990), EF1/ EF2 (O'Donnell et al. 1998), and 5F2/7cR (Reeb et al. 2004), respectively. The three gene sequences of four isolates showed 99.9 %-100% identities. The531 bp (ITS), 699 bp (TEF1), and 900 bp (RPB2) sequences of a representative isolate (JH-37) were deposited in GenBank with acce. nos. OR195729, OR195041 and OR195040, respectively. A phylogenetic tree was constructed using the concatenated three gene sequences of JH-37 and that of the related Fusarium species based on Maximum Likelihood (Fig.2). JH-37 was grouped together with the F. armeniacum strain CBS 485.94 (AB587001, GQ915501, GQ915485), and shared 99.9 % concatenated sequence identity. The three gene sequences of the strain JH-37 shared 100%, 99.85%, 99.89% identity to F. armeniacum strain CBS 485.94 using MEGA 7 software (Kuma et al. 2016) analysis, and with 94%, 95% and 100% coverage by BLAST analysis. The colony of JH-37 on PDA at 25°C for 5 days was white with yellow-brown pigmentation in the center (Fig. 1B-C). From 10-day-old cultures grown on Spezieller Nahrstoffarmer agar (SNA), macroconidia (n = 50) were falcate, slender, curved dorsiventrally, tapering towards both ends, 3 to 4 septate, and measured 24.2 to 50.0 × 2.6 to 4.2 μm. The microconidia (n = 50) were straight or slightly curved, septate 0 to 2, and measured 6.8 to 20.0× 2.1 to 3.7 μm (Fig.1D-F). These morphological characteristics were consistent with Fusarium spp. (Leslie and Summerell 2006). A pathogenicity test of JH-37 was performed on potted lily ('Tresor') under greenhouse conditions. Healthy lily bulbs were selected and one bulb was sown in soil of each pot. Inoculation was performed 60 days after sowing. Bulbs of the lily plants were wounded with needles and inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in the soil around bulb or an equal amount of sterilized water as a control. This experiment had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, and leaf wilt, similar to the original field symptoms, appeared on the inoculated plants but not on the controls (Fig.3). The same fungus was reisolated from the diseased plants, as identified based on morphology and molecular evidence, which confirmed the Koch's postulate. To our knowledge, this is the first report that F. armeniacum caused Fusarium wilt on Lilium spp. in China. Further, our result could help to develop effective disease management strategies against lily wilt disease.

First Report of Root and Bulb Rot Caused by Rhizoctonia solani AG-6 on Lanzhou Lily (Lilium davidii var. unicolor) in China.

Lanzhou lily (Lilium davidii var. unicolor) is the only famous sweet lily variety that has high edible, medicinal and ornamental value in China, which is mostly planted in the middle areas of Gansu Province in China. In recent years, severe yellowing and wilting of leaves, stem wilt, root and bulb rot symptoms were observed on Lanzhou lily in Qilihe District, Lanzhou, which has resulted in serious loss of bulb production. From June to August 2022, a survey of Lanzhou lily disease was carried out in Xiguoyuan and Weiling township of Qilihe District, Lanzhou. Typical symptoms of root and bulb rot were observed in Lanzhou lily fields. The disease incidence was estimated up to 30%. Fragments of symptomatic roots and bulbs were surface sterilized with 75% ethanol for 10 s, 2% sodium hypochlorite for 2 min, washed three times with sterilized distilled water, and then blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25 ± 1°C in darkness for 5 days and 2 isolates were purified by the single-tip culture. Colonies of the fungus were white initially, and then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 25 days of incubation at 25 ± 1°C in the dark. Genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences of strains QLH22LD01 and QLH22LD02 were deposited in GenBank (OR710804 and OR710805). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the two isolates within the R. solani AG-6 clade with high bootstrap support (100%). PCR analysis was performed with 21 primers specifically designed to detect individual anastomosis groups or anastomosis subgroups of R. solani (Carling et al., 2002; Misawa and Kurose, 2019; Misawa et al., 2020; Okubara et al., 2008). Among the 21 specific primer pairs, only AG-6 specific primer amplified the fungal DNA, indicating that the two isolates tested belonged to the R. solani AG-6. Therefore, these two strains were identified as R. solani AG-6. For pathogenicity tests, two isolates were grown individually on sterile wheat kernels at 25 ± 1°C for 14 days. Certified pathogen-free Lanzhou lily bulbs were grown in the plastic pot filled with the sterilized soil. Fifteen 2- week-old plants were inoculated by digging the soil and burying ten infested wheat kernels in the soil adjacent to the roots. Control plants were inoculated with sterile wheat kernels using the same procedure. All plants were placed in a greenhouse with a 12h/12h light/dark photoperiod at 15 to 30°C. Fifty days after inoculation, typical root and bulb rot symptoms developed on all inoculated plants, similar to symptoms observed in the field, whereas control plants remained symptomless. Pathogenicity test was performed three times with similar to symptoms observed in the field. Finally, the fungi were reisolated from the symptomatic plants and identified by molecular analysis as the isolates used for inoculation, thus fulfilling Koch's postulates. To our knowledge, this is the first confirmed report of R. solani AG-6 causing root and bulb rot on Lanzhou lily in China. Our findings improve knowledge about R. solani AGs occurring in Lanzhou lily fields in China. Due to serious damages caused by this disease in recent years in China, further studies should be conducted to investigate the diversity, prevalence, disease control measures and fungicide sensitivity of AGs distributed in the main Lanzhou lily-producing states in China.

First report of Fusarium equiseti causing bulb rot on lily (Lilium 'White planet' ) in China.

Lily (Lilium spp.) is one of the main ornamental plants grown in the world. In addition, bulbs of lily have been extensively used as edible and medicinal herbs in northern and eastern Asia, especially in China (Yu et al. 2015; China Pharmacopoeia Committee 2020; Tang et al. 2021). In August of 2021, a disease of stem and leaf rot was observed on lily cultivar 'White planet' with approximately 25% disease incidence in the greenhouse and fields at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China). The bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants. Infected bulbs were surface sterilized in 75% ethanol for 30 s, then in 2% sodium hypochlorite for 5 min, and rinsed three times with sterile distilled water. A 0.5×0.5 cm2 tissue piece was then placed on potato dextrose agar (PDA) medium and incubated at 25±1℃. After 5 days, the isolate was purified by single spore isolation technique. The singled-spored fungal colony was characterized by fluffy white aerial mycelia, and produced orange pigments with age. After seven days on Spezieller Nahrstoffarmer agar (SNA), conidia produced from simple lateral phialides. Macroconidia have pronounced dorsiventral curvature typical, significantly enlarged in the middle, a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 3 to 6 septate, measuring 18.71 to 43.01×2.89 to 5.56 μm with an average size of 26.98×3.90 μm (n=30). Microconidia were not observed. Typical verrucose thick chlamydospore with rough walls were profuse in chains or clumps, ellipsoidal to subglobose. These morphological characteristics were consistent with Fusarium spp. (Leslie et al. 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymeraseⅡsubunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2 and 5F2/7cR respectively and sequenced (White et al. 1990; Jiang et al. 2018; O'Donnell et al. 2007). Sequences were submitted to GenBank under accession numbers OM078499 (ITS), Accession OM638086 (TEF1-α) and OM638085 (RPB2). BLAST analysis showed that ITS, TEF1-α and RPB2 sequences shared 100%, 99.8%, 99.2% identity to F. equiseti (OM956073, KY081599, MW364892) in GenBank, respectively. In addition, ITS, TEF1-α and RPB2 sequences shared 100%, 99.53%, 100% identity with Fusarium lacertarum (LC7927, Fusarium incarnatum-equiseti species complex) in the Fusarium-ID database. Based on the morphological characteristics and molecular sequences, the isolates were identified as Fusarium equiseti. A pathogenicity test was performed on potted lily ('White planet') under greenhouse conditions (25±1℃ with a 16 h light and 8 h dark cycle). Three healthy lily bulbs were selected and one bulb was planted in each pot filled with sterilized soil. Each pot was inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in te soil around bulbs with a stem length of 3 cm, with an equal amount of sterilized water as a control. This test had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, like those observed in the greenhouse and fields, developed on the inoculated plants but not on the controls. The same fungus was consistently reisolated from the diseased plants. To our knowledge, this is the first report that F. equiseti caused bulb rot on Lilium in China. Our result should help with future monitoring and control of lily wilt disease.

Phenotyping cowpea accessions at the seedling stage for drought tolerance in controlled environments

Abstract One of the most important screening techniques used in cowpea selection for drought tolerance is screening at the seedling stage. The objective of this study was to phenotype 60 cowpea genotypes for seedling drought tolerance in screen houses (glasshouse and greenhouse). Principal component analysis revealed that of the 14 variables, the first 4 expressed more than 1 eigenvalue. Data showed that PC1, PC2, and PC3 contributed 39.3, 15.2, and 10%, respectively, with 64.68% total variation. A PCA plot and biplot showed that the number of pods (NP), seeds per pod (SP), survival count (SC), pod weight (PWT), and stem wilting in week 1 (WWK1) had the most significant contributions to genetic variability to drought tolerance and to yield after stress imposition based on the PCA, biplot, and cluster plot, the accessions IT 07-292-10, IT 07-274-2-9, IT90K-59, 835-911, RV 343, and IT 95K-2017-15 had the maximum variability in terms of NP, SP, SC, PWT, and WWK1 after drought imposition. Cowpea accessions 835-911, IT 07-292-10, RV 344, Kangorongondo, and IT 90K-59 were the major individuals that contributed mainly to domain information model (DIM) 1 and 2. The accessions that contributed the least were IT 89KD288, Chibundi mavara, and TVU12746. Thirty-six cowpea accessions from both screen houses were tolerant to drought, 15 were moderately tolerant, while 23 were susceptible. The findings of the study provided a useful tool for screening and determining drought-tolerant and susceptible accessions at the seedling stage.

First Report of Sea Buckthorn Stem Wilt Caused by Fusarium proliferatum in Liaoning, China.

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O'Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O'Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 - 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 - 13.6 × 2.2 - 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.

Common problems of <i>Rhododendron</i> L. in the Central Botanical Garden of the National Academy of Sciences of Belarus

Rhododendrons are some of the most attractive flowering shrubs in the landscape. The article presents the results of studying the common problems of Rhododendron collection in the Central Botanical Garden of the National Academy of Sciences of Belarus, conducted in 2012-2020. There are several diseases and pests that commonly occur on rhododendrons every year. These include powdery mildew (Erysiphe azaleae), fungal leaf spots (Pestalotiopsis rhododendri, Colletotrichum gloeosporioides, Alternaria spp., Sphaerulina azaleae, Phyllosticta concentrica, Phyllosticta rhododendricola), black vine weevil (Otiorhynchus sulcatus), garden foliage beetle (Phyllopertha horticola), click beetle (Hemicrepidius niger), red spider mite (Tetranychus urticae), physiological leaf spots, oedema, winter injury, chlorosis. Less common on rhododendrons are stem diseases (Botryosphaeria dothidea, Cadophora spp., Hymenoscyphus spp.), wilt (Fusarium oxysporum), bud blight (Seifertia azaleae), leaf and flower gall (Exobasidium rhododendri), rhododendron lacebug (Stephanitis rhododendri), greenhouse whitefly (Trialeurodes vaporariorum). There were identified that all evergreen and half evergreen rhododendrons have some resistance to powdery mildew, deciduous rhododendrons are more resistant to fungal leaf spots than evergreens. Damage to rhododendrons by insect pests usually does not cause significant harm but heavy feeding results in an unsightly plants.

First report of Stenotrophomonas maltophilia causing root soft rot of Sanqi (Panax notoginseng) in China.

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a traditional Chinese medicinal plant with a long planting cycle of 2-3 years that makes it vulnerable to root diseases caused by several pathogens, including Fusarium solani, Alternaria panax, Phytophthoracactorum, and Pseudomonas sp. In April 2019, root soft rot samples of Sanqi were collected from a plantation site in Songming, southwest of China. Typical symptoms included root softening and necrosis, yellow leaf, and stem wilting. Ten diseased roots samples were collected and sterilized with 0.1% HgCl2 for 1 min, 75% ethanol for 2min, and then rinsed thrice with sterile water. Sterilized roots were cut into small pieces of 5 × 5 mm and cultured on the nutrient agar (NA) medium for 48 h at 28°C. From the root cultures, a total of thirteen bacterial strains were obtained. Three strains, SM 2-5, SM 2-13, and SM 2-14 were selected for further study. These three strains were gram-negative, short rod-shaped (1～2×0.5～1μm), non-spore-forming and had polar tufted flagella as observed under a transmission electron microscope (TEM). Also, the strains were positive for oxidase, beta-galactosidase, arginine dihydrolase, and lysine decarboxylase while negative for amylase and urease tested by biochemical methods (Wang 2017). To further determine the pathogenic species, genomic DNA of these three strains was extracted using a Genomic DNA Kit (Tsing Ke, Beijing, China), to PCR amplify 16S rDNA using universal primers 27F/1492R (Wang et al. 2017). Also, S. maltophilia 23S rDNA specific primers SM1/SM4 (Whitby et al. 2000) were used for PCR amplification to confirm the species. 16S rDNA sequence analysis showed that SM 2-5 (GenBank Accession No. MW555227), SM 2-13 (GenBank Accession No. MW555228), and SM 2-14 (GenBank Accession No. MW555229) shared the highest identity (>99.9%) with the S. maltophilia strains (GenBank Accession No. MT323142, MH669295, MN826555). Furthermore, 23S rDNA sequence analysis of SM 2-5 (GenBank Accession No. MZ707732), SM 2-13 (GenBank Accession No. MZ645941) and SM 2-14 (GenBank Accession No. MZ707733) revealed their high identity (>99.8%) with the S. maltophilia species. 16S and 23S rDNA phylogenetic analysis (Mega6.06) using the neighbor-joining (NJ) method with 1,000 bootstrap replicates revealed the three strains clustering with the other S. maltophilia strains. Therefore, based on morphology, metabolic profile, and sequence analysis, the three strains were identified as Stenotrophomonas maltophilia. To test pathogenicity, the strains were grown in the nutrient broth (NB) medium for 48h at 28°C until bacterial suspension reached to OD600≈1.0 (2.0×109CFU/mL). Then, healthy roots of one-year-old Sanqi plants, pre-washed with sterilized water and -poked with a sterilized needle, were soaked in bacterial suspension (2.0×109CFU/mL) of the three strains separately for inoculation 10min. Sterilized water treatment was used as a control. Subsequently, bacteria-inoculated plants were planted in sterile soil pots and cultured in a greenhouse at 28°C with shading rate of 70%. Each treatment group included 3 plants with 3 replicates. Ten days post inoculation, symptoms similar to the ones in natural conditions were observed in the bacteria-inoculated plants. Based on the disease index (Li et al. 2020), we found that among the three strains, SM 2-13 displayed the highest virulence, while no symptoms were observed in the control plants. The same bacterial strains were re-isolated from these inoculated roots and identified by the methods described above. Previous studies showed that some Stenotrophomonas species cause plant diseases such as rice white stripe (Singh et al. 2001), strawberry leaf black spot (Wang et al. 2017), Cyclobalanopsis patelliformis leaf spot (Bian et al. 2020), and Jatropha curcas L. seed borne and stem necrosis (Wang et al. 2018). To our knowledge, this is the first report confirming Stenotrophomonas maltophilia causing root soft rot of Panax notoginseng in China.

First Report of Sea buckthorn Stem Wilt Caused by Fusarium sporotrichioides in Gansu, China.

Sea buckthorn (Hippophae rhamnoides) is an important deciduous shrub for fruit and ecological restoration in arid and semi-arid regions of China. Twelve Chinese and Russian cultivars (cv. Shenqiuhong, eshi01, ... eshi11) were planted about 1.6 acre area in a seedling nursery, located in Qingyang City of Gansu province in northwest China, where high mortality (more than 70%) of sea buckthorn was observed in late July 2019. Symptoms consisted of massive chlorosis, drooping leaves and dried-up stems on 5-year-old trees. Pieces of tree roots and stems with irregular light-brown discoloration in the xylem vessels were selected. Small pieces of discolored tissue were surface disinfested (1 min in 1% sodium hypochlorite, followed by three rinses with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues. Colonies on PDA grew rapidly. Dense mycelia were pinky-white initially, and became carmine red color with age on the undersurface of the plate. Macroconidia were moderately curved, 3 to 5 marked septa, hyaline, thick walled, and measuring 27.8± 3.6 µm × 4.8 ± 0.5 µm (n = 30). Microconidia were abundant, pear-shaped, ellipsoid to fusoid, often with a papilla at the base, and 8.4 ± 2.2 µm ×3.1 ± 0.3 µm (n = 30). Genomic DNA was extracted for amplification and sequencing of the internal transcribed spacer region (ITS1 and ITS4 primers) (White et al. 1990) of the ribosomal DNA (Accession Nos. MN160235 to MN160238) and translation elongation factor-1 alpha (EF1 and EF2 primers, accession Nos. MN429075 to MN429078) (O'Donnell et al. 1998). The sequences revealed 99% similarity to the sequences of the ITS (AY188917), and 100% identity with EF1-α (JF740808) regions of Fusarium sporotrichioides. Based on morphological and molecular characteristics, the fungus was identified as F. sporotrichioides (Leslie and Summerell 2006). Koch's postulates were fulfilled on healthy, potted 1-year-old sea buckthorn seedings using two isolates in a greenhouse at 25 °C, 90% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedings were inoculated with PDA plugs as controls. Seven to ten days after inoculation, typical symptoms of dark-brown necrotic lesions on chlorotic leaf margins were observed. About 2 weeks after inoculation, the inoculated stems were gradually dry up, accompanied by withering and fallen leaves. Control plants remained asymptomatic. Pathogens were successfully isolated from the inoculated stems again, exhibiting morphological characteristics identical to those of F. sporotrichioides. Previous papers reported F. sporotrichioides as a common pathogen caused lavender wilt (Cosic et al. 2012), foliar spots on forage corn (Moya-Elizondo et al. 2013) and maize ear rot (Wang et al. 2019). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. sporotrichioides on several Chinese and Russian cultivars in Gansu province of China. In Heilongjiang province, the same disease was reported in 2010 (Song et al. 2010), nearly 30 longitudes away from Gansu province. Therefore, this disease appears to be a serious risk for future sea buckthorn production.

First Report of Blackleg DiseaseCaused by Epicoccum sorghinumon on Lavender (Lavandula stoechas) in China.

Lavender (Lavandula angustifolia Mill) is an ornamental plant and worldwidely grown for its aromatic and pharmacological qualities. In June 2020, the symptoms of blackleg disease on lavender plants were observed, with more than 50% incidence in Chaohu city (117°38'19.12″N, 31°47'18.94″W) of Anhui Province, China. The disease symptoms progressed from stem wilt and necrosis to prolonged necrosis and bending of leaves, and all infected lavender plants died eventually. Ten necrotic stem lesions werecollectedfrom ten independent plants for the isolation of pathogen. All samples were washed in 70% ethanol for 1 minute, rinsed twice in sterile distilled water and placed on water agar (WA) plates containing 30 mg/liter of kanamycin. All 16 fungal isolates were transferred onto potato dextrose agar (PDA) and incubated at 26°C for 5 days, and all fungal colonies were isolated consistently, which produced redish-gray mycelium at 26°C with a 12-h photoperiod on PDA media. They developed black pycnidia with abundant hyaline, unicellular, oval shaped conidia (4.5 to 5.9 × 2.1 to 2.5 μm) after 14 days. DNA was extracted (10-day-old culture) using the Fungal DNA Mini Kit (Omega Bio-tek, China), according to the manufacturer's protocol. The internal transcribed spacer (ITS), beta-tubulin (β-tub) and translation elongation factor 1-alpha (tef1-α) genes of three isolates were amplified using the primers: ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The ITS(MT883331), β-tub(MT896891) andtef1-α (MT874165) genes were sequenced and analyzed through BLASTn. The ITS sequence showed 99.81% with Epicoccum sorghinum (GenBank Accession No. MK020690.1). The β-tub and tef1-α showed 100% homology with Epicoccum sorghinum (GenBank AccessionMN554062.1 and MN512426.1), respectively. To complete Koch's postulates, pathogenicity tests were performed by spraying the fungal spore suspension (1×105 CFU/ml) prepared from 14-day-old cultures onto needling wounded stems of 1-year-old potted healthy L. angustifolia plants. The healthy plants were sprayed with sterilized water onto needling wounded stems served as negative control. Wilting and stem necrosis were observed 5 days afterinoculation and incubation in a growth chamber at 26°C, with a 12-h photoperiod. All fungal infected plants died after 10 days, while, the control plants remained healthy. The fungus was re-isolated from the lesions of the inoculated plants and verified. Based on morphological characteristics, sequence analysis and pathogenicity test, the pathogen was identified as E. sorghinum. The pathogen has been observed previously on many plants such as tea (Bao et al. 2019) and taro (Liu et al. 2018), in China. To our knowledge, this is the first report of E. sorghinum causing blackleg disease of lavender in China and worldwide.

Virus-Induced Gene Silencing of SlWRKY79 Attenuates Salt Tolerance in Tomato Plants

Previous studies have shown that WRKY transcription factors play important roles in abiotic stress responses. Thus, virus-induced gene silencing (VIGS) was used to identify the function of SlWRKY79 in the salt tolerance of tomato plants by downregulating the expression of the SlWRKY79 gene. Under the same salt treatment conditions, the SlWRKY79-silenced plants showed faster stem wilting and more severe leaf shrinkage than the control plants, and the bending degree of the stem of the SlWRKY79-silenced plants was also greater than that of the control plants. Physiological analyses showed that considerably higher levels of hydrogen peroxide (H2O2), superoxide anion (O2−), and abscisic acid (ABA) accumulated in the leaves of the SlWRKY79-silenced plants than in those of the controls after salt treatment. Taken together, our results suggested that SlWRKY79 plays a positive regulatory role in salt tolerance in tomato plants.

First report of Fusarium fujikuroi causing bulb rot on Lilium lancifolium in China.

First report of Fusarium fujikuroi causing bulb rot on Lilium lancifolium in China.

Establishment of the Causal Agent(s) of Spike Shedding and Stem Wilting of Pepper (Piper nigrum L.) in Morogoro District, Tanzania

Pepper (Piper nigrum L.) serves as a source of income to smallholder spice farmers in Morogoro district of Tanzania. Recently, spike shedding and stem wilting of pepper plants were reported to cause about 30% yield loss of the crop. This study was undertaken to identify the causal agent(s) of the problems. Three pepper gardens in each of the four hamlets (Nyange, Doga, Tandai, and Lukenge) in Tandai village (Kinole ward) were surveyed, and plant tissue and soil samples were collected for laboratory analysis. An experiment was laid out in a randomized complete block design with three replications. Pepper cultivars, Babu kubwa, Babu ndogo, Babu kati, and Ismailia grown in the study area, were used as treatments for evaluation of the prevalence of spike shedding and stem wilting. Treatments mean separation was conducted based on Duncan’s multiple range test atp=0.05. Regression (R2) and simple correlation (r) analyses between stem wilting (incidence and severity) and termite pest infestation were performed. The identified pathogen in most of the soil and plant tissue samples was Fusarium oxysporum. The nutrients, N, P, Mg, and Cu, concentration in soil and leaf samples was below the optimal ranges. Termite attacks were significantly high in some farmer’s gardens where up to 50.5% of the pepper plants was damaged. Hence, a significant positive correlation was observed between termite pest infestation in gardens and both incidence (r = 0.881, R2 = 0.74,p<0.001) and severity of stem wilting (r = 0.918, R2 = 0.84,p<0.001) of pepper plants. Most of the termite attacks and damages (70%) were observed on pepper plants supported on silver oak, unlike those trained on Jatropha. A higher percentage of undeveloped berries (36%) and intermediate-size berries (34%) was recorded on cultivars “Babu kati” and “Babu kubwa,” respectively. Multiple factors have been associated to be the causes of spike shedding and stem wilting of pepper, which could be used to develop appropriate control solutions useful to farmers. Confirmation of F. oxysporum pathogenicity is recommended. A comprehensive study is suggested on the repellent or lethal activities of Jatropha against F. oxysporum and termites. Furthermore, studies through field experiments are needed to generate site-specific soil nutrient improvement recommendations and design an integrated approach to control F. oxysporum and termites and to determine existing alternative host plants for these pests.

Extent of spike shedding and stem wilting of pepper (Piper nigrum L.) in Morogoro District, Tanzania

BackgroundPepper (Piper nigrum L.) is an important spice produced by small-scale farmers in Morogoro district of Tanzania for both local and export markets. Recently, farmers have reported a decline in crop productivity due to spike shedding and stem wilting of pepper plants. The study was conducted to obtain baseline information on the extent of the disorders.MethodsAn altitudinal transect survey was conducted in Morogoro district from October to November 2018. Data were collected from 216 pepper farmers through a questionnaire, focus group discussions, and field observations. Chi square tests (α = 0.05) were used to compare differences between gender, pepper cultivars by wards and yield, spike shedding and stem wilting by pepper cultivars and plant species used to support pepper vines. Fisher’s exact test at (α = 0.05) was used to compare the effects of production constraints and altitude. A regression analysis was performed to explore the relationship between altitude and the incidence of spike shedding and stem wilting.ResultsThe majority of interviewees (69%) had observed spike shedding and stem wilting of pepper on their farms for > 3 years. Spike shedding was more prominent at 300 meters above sea level (masl, 85.7%), while stem wilting was reported by most (66.7%) farmers at 457 masl. The most affected pepper cultivar was reported to be “Babu kati” in Mtombozi (19.2% of respondents) and “Ismailia” in Mkuyuni and Kibogwa (28.9% of respondents) wards, while in Kibungo ward cultivar “Babu ndogo” (85.7% of respondents) and in Konde ward cultivar “Babu kubwa” (53.8% of respondents) were most affected. Approximately 53% of respondents reporting the disorders used Jatropha to support the pepper vines, and 93% of the farmers reported the disorders to occur during the dry season. A greater proportion of the interviewed farmers (92.1%) reported applying no measures to control spike shedding and stem wilting of pepper.ConclusionsIncidence of spike shedding and stem wilting was higher at a low altitude compared to high altitude areas. The magnitude of the disorders differed among pepper cultivars regardless of support plants species used. Poor adoption of key agronomic practices and lack of any information on control measures may be contributing to the high incidence of the disorders in the study area. The study demonstrates the extent of the problem, and provides a baseline for identifying the causal agent(s) of spike shedding and stem wilting, which in turn will allow appropriate control measures to be selected.

First report of stem wilt caused by Fusarium oxysporum f. sp. opuntiarum on Sulcorebutia rauschii in Italy

First report of stem wilt caused by Fusarium oxysporum f. sp. opuntiarum on Sulcorebutia rauschii in Italy

Agrobacterium tumefaciens-mediated transformation of a hevein-like gene into asparagus leads to stem wilt resistance.

Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 μmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.

Occurrence of Maize Ear Rot Caused by Fusarium fujikuroi in China

Maize (Zea mays L.) is one of the most important crops in China. Ear rot is a serious disease that often leads to a decline in maize yield and quality and is a hazard to human and livestock health owing to potential mycotoxin contamination of grain. The incidence of maize ear rot usually ranges from 20 to 40% at different fields, even reaching as high as 70% at certain plots in Chongqing and surrounding areas (Zhou et al. 2018). In August 2016, a survey to determine population composition and distribution of Fusarium spp. causing maize ear rot was conducted in 22 counties in Chongqing. Maize ears with white, pink, or salmon-colored mold were collected from fields for further analysis. Symptomatic kernels were soaked in 2% sodium hypochlorite solution for 3 min, rinsed with sterile water three times, placed on acidified potato dextrose agar (PDA), and incubated at 26 ± 1°C for 3 to 5 days. The newly grown-out fungal colonies displaying morphological characteristics of Fusarium spp. were transferred onto fresh PDA and Spezieller Nahrstoffarmer agar and purified by the single-spore isolation method (Yang et al. 2008). Fusarium spp. were identified based on morphological characteristics (Leslie and Summerell 2006) and sequence analysis of the translation elongation factor-1α (TEF-1α), β-tubulin, and mitochondrial small subunit (mtSSU) genes (Jiang et al. 2018). The predominant Fusarium spp. were Fusarium verticillioides, F. proliferatum, and F. meridionale, which were the main causal agents of maize ear rot in these areas. In addition, morphological characteristics of several isolates from Beibei, Jiangjin, Xiushan, Rongchang, Kaixian, and Wanzhou Districts were found to be identical to those of Fusarium fujikuroi Nirenberg, which grouped into the Gibberella fujikuroi species complex. Colonies on PDA produced abundant white aerial mycelia initially, but the mycelia became pale pinkish and dark violet with age. Microconidia were abundant and small in size (4.9 to 11.3 × 2.2 to 3.2 µm), colorless, oval to club shaped, and 0- to 1-septate. Macroconidia were slender, almost straight, sickle shaped, medium length, with three to five septa (17.1 to 39.2 × 2.6 to 3.3 µm). No chlamydospores were observed. Identity of the fungus was also confirmed by sequence comparison of the partial TEF-1α, β-tubulin, and mtSSU genes of two representative isolates. The resulting sequences of the TEF-1α, β-tubulin, and mtSSU genes showed 100, 100, and 99% sequence identity to those of F. fujikuroi (GenBank accession nos. MH263736.1, MH263737.1, and MF984420.1, respectively). DNA sequences of partial TEF-1α, β-tubulin, and mtSSU genes from two representative isolates (FCQD90 and FCQD97) were deposited in GenBank under accession numbers MK328875 and MK328876, MK385626 and MK385627, and MK385628 and MK385629, respectively. A pathogenicity test was performed on maize inbred lines B73 and Ye 107. Four days after silk emergence, 2 ml of conidial suspension (1 × 10⁶ conidia /ml) of each isolate was injected into each of 10 maize ears through the silk channel. Control plants were inoculated with sterile distilled water. The typical Fusarium ear rot symptom (white or white-pink mold) was observed on inoculated ears and the infected kernels. No symptom developed on the water-inoculated controls. F. fujikuroi was reisolated from the symptomatic ears but not from the control. F. fujikuroi was previously reported associated with seedling damping-off on soybean, stem wilt of Canna edulis, and root rot of Reineckia carnea (Jiang et al. 2018; Pedrozo et al. 2015; Sun et al. 2018). To our knowledge, this is the first report of F. fujikuroi causing maize ear rot in China, and this disease should be paid sufficient attention owing to a serious risk of mycotoxin contamination in maize.

Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle

BackgroundPectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).ResultsThirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.ConclusionsThis study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.