Infected Bulbs Research Articles

First Report of Trichoderma hamatum Causing Postharvest Bulb Rot on Lilium davidii var. willmottiae in China.

Lilium davidii var. willmottiae, known as Lanzhou lily, is a famous edible crop that is mostly distributed in the middle area of Gansu Province in China. In the winter of 2019, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Grassland, Flowers and Ecology (39°57'55.984" N, 116°20'8.124" E), Beijing Academy of Agriculture and Forestry Sciences, at an incidence of nearly 50%. The decayed bulb (Fig.1a)was washed under tap water and surface disinfested with 75% ethanol for 1 min, followed by 2.5% sodium hypochlorite for 5 min, and washed with sterile distilled water three times. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Thirteen dominant pure fungal isolates with the same morphological characteristics were obtained by the hyphal-tip method. Three representative isolates LZ-8, LZ-9-2 and LZ-10 were chosen for phylogenetic analyses. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1a), and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RPB2-5F2/RPB2-7cR (O'Donnell et al. 2022), respectively. BLAST analysis showed that the ITS,TEF-1a, and RPB2 sequences of the isolates LZ-8 (GenBank accession nos. PP422096, PP447248, and PP447251), LZ-9-2 (GenBank accession nos. PP422098, PP447249, and PP447252) and LZ-10 (GenBank accession nos. PP422099, PP447250, and PP447253) had 99.27 to 99.71% identity with multiple GenBank sequences of Trichoderma hamatum, and the three DNA fragments of the three isolates showed 100% sequence identity. A phylogenetic tree based on concatenated sequences of the three genes using maximum -likelihood analyses revealed that the three isolates LZ-8, LZ-9-2 and LZ-10 were in the same clade with T. hamatum strains (Fig.2). One representative isolate, LZ-10, was chosen for morphological studies and test of the pathogenicity. The colony of LZ-10 on PDA appeared white with cotton-shaped aerial hyphae early, which later turned light green to green and formed concentric rings (Fig.1d-1f). At the end of conidiophores, three to six pear-shaped branches were irregularly gathered(Fig.1h). Conidia were ellipsoid with the size of 3.1 to 4.4 × 2.2 to 3.1 µm (n =20) (Fig.1g). These morphological characteristics were consistent with the description of Trichoderma hamatum. (Kamala et al. 2015, Han et al. 2017).To test pathogenicity, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 6 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers(Fig.1b-1c) consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, meanwhile the uninoculated lily bulbs remained symptomless. Trichoderma hamatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Trichoderma hamatum in China. This study will contribute to a better understanding and controlling of this postharvest disease in Lilium davidii var. willmottiae.

First Report of Erwinia aphidicola Causing Bulb Rot of Onion in Chile.

During the 2021-22 and 2022-23 seasons (December to February), onion plants (Allium cepa L.) showing decay, leaf blight, chlorosis and water soak lesions were collected in Central Chile. Five symptomatic plants were sampled from 20 different onion fields. Brown rot of the external scales was observed in bulbs from two fields: one planted with the cv. Campero (20 ha; O'Higgins Region), and another with cv. Marenge (2 ha; Metropolitan Region). The disease incidence in these fields ranged from 2% to 5%. Isolations were carried out from symptomatic leaves and bulbs from these fields on King's B medium, resulting in small white colonies with smooth margin. Three isolates were selected, two from first field (QCJ3A & QCJ2B), and one from second field (EPB1). A preliminary identification based on 16S rRNA sequences was conducted. BLAST analyses of strains QCJ3A, QCJ2B and EPB1 (GenBank Accession No. PP345601 to PP345603) against the NCBI Database resulted in a match with strains (GenBank Accession No. ON255770.1 and ON255825.1) isolated from infected bulbs in Texas, USA identified as Erwinia spp. (Khanal et al. 2023), with 100% coverage and 100% identity (707 bp out of 707). To evaluate the pathogenicity of these three strains, onion bulbs were inoculated (Guajardo et al. 2023). Toothpicks previously immersed in a bacterial suspension at ~ 108 colony forming units (CFU)/mL were pricked at a 4 cm depth into the shoulders of onion bulbs bought from commercial store and incubated at room temperature. Bulbs inoculated with sterile water served as negative control. A known onion bulb rotting bacterial strain of Dickeya sp. was used as a positive control. At the end of the incubation period (20 days), bulbs were opened longitudinally across their inoculation site, showing that the external scales had a brown color. Negative control remained asymptomatic. Strains were re-isolated from damaged tissue and identified as Erwinia sp. This assay was repeated three times with the same results. For further identification, genomic DNA extraction was carried out using the Blood & Cell Culture DNA Kit (Qiagen), and genome sequencing was performed in the Illumina HiSeq 2500 platform. The Whole Genome Shotgun project for strains QCJ3A, QCJ2B and EPB1 have been deposited at DDBJ/ENA/GenBank under the accession JBANEI010000000, JBANEJ010000000 and JBANEK010000000. The average nucleotide identity (ANI) values were 99.6% (EPB1), 98.2% (QCJ2B), and 99.6% (QCJ3A) and DNA-DNA hybridization (dDDH) values were 96.9% (EPB1), 83.7% (QCJ2B), and 97.1% (QCJ3A), when compared with the type strain Erwinia aphidicola JCM 21238 (GenBank accession No. GCF_014773485.1). The three strains were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Erwinia aphidicola has been previously described causing diseases in common bean (Phaseolus vulgaris) and pea (Pisum sativum), in Spain (Santos et al. 2009) and in pepper (Capsicum annuum) in China (Luo et al. 2018). Its close relative E. persicina has been reported causing bulb rot in onion in Korea (Cho et al. 2019) and garlic in Europe (Galvez et al. 2015). To our knowledge, this is the first report of E. aphidicola causing a bulb rot of onion in Chile. Although the distribution and prevalence of this bacterium in Chilean agroecosystems is not known, it can be a potential cause of losses in onions and other crops such as beans, peas, and peppers. Additional studies should be conducted to determine the host range of Chilean Erwinia aphidicola strains.

Effects of Different Growing Medium on Obtaining Bulblets from Bulb Scales in Oriental Lilium 'Siberia' Cultivar

This study was carried out to determine the best medium for obtaining bulblets in the Oriental Lilium 'Siberia' plant using bulb scales and different media. In the research, six different growing mediums were used as growing media: soil (control), peat, soil + peat, perlite, perlite + peat, and peat + sand. In the experiment, the number of bulblets, bulblet width, bulblet length, number of healthy and infected bulb scales, and the time it took for the first bulbs to formation were measured. As a result of the study, the highest number of bulblets (110) were obtained from peat + sand. The growing medium with the highest values of bulblet width (11.38 cm) and bulblet length (16.27 cm) was determined to be peat. The highest number of healthy bulb scales (54) was obtained from perlite medium, and the highest number of infected bulb scales (20) was obtained from soil medium. In terms of the formation time of the first bulblets, perlite medium (15 days) was the growing medium where the earliest bulbs were obtained, and the latest bulblets were obtained from control (soil) and soil + peat (40 days). In general, the quality of bulblets obtained from peat medium was better than that from other mediums. In this regard, peat growing medium is recommended to obtain quality bulblets, and peat + sand is recommended if the number of bulblets is desired to be high.

First Report of Fusarium proliferatum Causing Bulb Rot on Lilium davidii var. willmottiae in China.

Lilium davidii var. willmottiae, known as Lanzhou lily, is widely cultivated in China for its edible bulbs. In July 2023, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China), at an incidence of nearly 70%. The surface of the lily scales had dark water-stained spots, after the development of which the color gradually darkened, the bulbs became soft, accompanied by a pungent smell. Finally, the whole bulb became ruined and rotten, and there were thick mycelium layers on the bulbs. The infected bulbs were washed with clean water, sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 5 min, and then rinsed three times with sterile distilled water. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 fungal isolates were obtained, all of which exhibited typical Fusarium characteristics. The colonies were white to pink with white to cream-colored aerial mycelia. After 10 to 15 days of incubation, the macroconidia (n = 50) were hyaline, relatively slender with a curve, three to five septate, and 8.73 to 33.24 × 2.16 to 4.19 μm in length. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 4.04 to 8.48 × 1.24 to 2.65 μm. These morphological characteristics were similar to those described for Fusarium proliferatum (Leslie and Summerell 2006). For molecular identification, a cetyltrimethylammonium bromide (CTAB) protocol was used to extract total genomic DNA (O'Donnell et al., 1998), after which the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymerase Ⅱ subunit 2 (RPB2) genes were amplified using the universal primers ITS1/ITS4, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively, and subsequently sequenced (White et al., 1990; O'Donnell et al., 1998; Liu et al., 1999; Reeb et al., 2004; O'Donnell et al., 2007; Jiang et al., 2018). The sequences of a representative isolate (CAAS01) were analyzed and submitted to GenBank under accession numbers OR554007 (ITS), OR594233 (TEF1-α) and OR603932 (RPB2). A BLAST analysis revealed that the sequences of the ITS, TEF1-α, and RPB2 genes shared 100%, 100%, and 100% identity, respectively, with those of Fusarium proliferatum (MT466521.1, MK952792.1, and LT841266.1) in GenBank. In addition, the ITS, TEF1-α and RPB2 sequences shared 100%, 100%, and 100% identity with those of Fusarium annulatum (LC13675, the Fusarium fujikuroi species complex; previously known as the Gibberella fujikuroi species complex) in the Fusarium-ID database. Fusarium proliferatum, whose common synonyms are Gibberella fujikuroi mating population D and Gibberella fujikuroi var. intermedia, is the anamorphic form of the Gibberella fujikuroi complex that belongs to the Nectriaceae family. A phylogenetic tree was constructed based on the combined TEF1-α and RPB2 sequences of CAAS01 and other Fusarium isolates, revealing that CAAS01 was grouped with Fusarium proliferatum. Based on sequence alignment and phylogenetic analysis, the isolate was identified as Fusarium proliferatum. To determine the pathogenicity of the isolated fungi, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 7 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, while the uninoculated lily bulbs remained symptomless. Fusarium proliferatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Fusarium proliferatum in China. This study will contribute to the development of management strategies for this postharvest disease in Lilium davidii var. willmottiae.

Identification and Aggressiveness of Fusarium Species Associated with Onion Bulb (Allium cepa L.) during Storage.

Plant pathogens present a major challenge to crop production, leading to decreased yield and quality during growth and storage. During long-term storage, healthy onions can develop diseases from latent pathogen infections. This poses a challenge for onion growers because infected bulbs without visible symptoms can lead to significant crop losses during the growing season. In this study, we aimed to isolate and identify Fusarium species from yellow onion bulbs (Allium cepa L.) that developed disease symptoms during storage. The aggressiveness of these strains against onion bulbs and seedlings was also evaluated. The isolated strains were further subjected to morphological and molecular differentiation. The results revealed that all 16 isolated strains belonged to the Fusarium complex species incarnatum-equiseti and Fusarium fujikuroi, namely, F. proliferatum (98%), F. oxysporum (1%), and Fusarium sp. (1%). Koch's postulate analysis of isolated strains revealed varying aggressiveness on onion bulbs and plants depending on fungal species. Disease symptoms developed more slowly on plants than on onion bulb plants according to Koch's postulates. Moreover, the results revealed that Fusarium strains that can infect onion plants were less pathogenic to onion bulbs and vice versa. In addition, three isolates were found to be non-pathogenic to onions. Furthermore, the in vitro control of Fusarium species through Bacillus velezensis KS04-AU and Streptomyces albidoflavus MGMM6 showed high potential for controlling the growth of these pathogenic fungi. These results may contribute to the development of environmentally friendly approaches for controlling onion spoilage caused by pathogens during storage.

Different Preharvest Diseases in Garlic and Their Eco-Friendly Management Strategies.

garlic reproduces mainly through clove planting, as sexual reproduction via seeds is uncommon. Growers encounter challenges with pathogens due to the larger size and vegetative nature of seed cloves, as well as the storage conditions conducive to fungal growth. Some Phyto-pathogenic fungi, previously unrecognized as garlic infections, can remain latent within bulb tissues long after harvest. Although outwardly healthy, these infected bulbs may develop rot under specific conditions. planting diseased seed cloves can contaminate field soil, with some fungal and bacterial infections persisting for extended periods. The substantial size of seed cloves makes complete eradication of deeply ingrained infections difficult, despite the use of systemic fungicides during the preplanting and postharvest phases. Additionally, viruses, resistant to fungicides, persist in vegetative material. They are prevalent in much of the garlic used for planting, and their host vectors are difficult to eliminate. To address these challenges, tissue-culture techniques are increasingly employed to produce disease-free planting stock. Key scientific concepts of the review: garlic faces a concealed spectrum of diseases that pose a global challenge, encompassing fungal threats like Fusarium's vascular wilt and Alternaria's moldy rot, bacterial blights, and the elusive garlic yellow stripe virus. The struggle to eliminate deeply ingrained infections is exacerbated by the substantial size of seed cloves. Moreover, viruses persist in garlic seeds, spreading through carrier vectors, and remain unaffected by fungicides. This review emphasizes eco-friendly strategies to address these challenges, focusing on preventive measures, biocontrol agents, and plant extracts. Tissue-culture techniques emerge as a promising solution for generating disease-free garlic planting material. The review advocates for ongoing research to ensure sustainable garlic cultivation, recognizing the imperative of safeguarding this culinary staple from an array of fungal and viral threats.

Study on Italian Onion Cultivars/Ecotypes towards Onion Yellow Dwarf Virus (OYDV) Infection

The onion yellow dwarf virus (OYDV) represents a limiting biotic stress in onion (Allium cepa L.); little information is available regarding resistant varieties. In Italy, onion production is limited but represented by a wide diversity of ecotypes. A two-year trial was carried out to test the OYDV-susceptibility/tolerance of different Italian onion cultivars by rating symptom severity and plant growth parameters and assessing post-harvest secondary infections. The cultivar and ecotypes included in the study were characterized by simple sequence repeats (SSR) analysis, and the expression analysis of two genes (Eukaryotic translation initiation factors, EIFs) involved in potyvirus replication was also performed. Two susceptible and one tolerant cultivar were identified based on symptom expression and virus impact on plants. Although differences in growth parameters were limited to the first-year trial, the infection was correlated to a higher incidence of secondary infections in post-harvest, with altered water balance in infected bulbs. This correlation was also demonstrated during the long-term storage of bulbs. SSR analysis identified different clusters and only one gene isoform (EIF4eiso1) showed different expression levels in the OYDV/onion pathosystem. In conclusion, this study defines the genetic profile of Italian onion cultivars and provides evidence on susceptibility/tolerance features which will be useful in the future for the identification of viral resistance traits in onion.

Potential of Compost Enriched with Bacillus velezensis B-27 and Bacillus cereus RC76 for the Management of Twisted Disease on Shallots

Shallot (Allium cepa var. aggregatum.) is a horticultural plant that is widely consumed in the world. However, the productivity of shallots in Indonesia is still relatively low, if compared to the actual optimum production potential of shallot. Shallot cultivation in Indonesia often experiences many problems. One of the problems is twisted diseases caused by Fusarium sp. This research aimed to study the effect of the application of organic material enriched with Bacillus in suppressing the development of twisted disease of shallot. This study was arranged in Randomized Complete Block Design (RCBD) with 5 treatments namely (A) compost + Bacillus velezensis isolate B-27, (B) compost + Bacillus cereus isolate RC76, (C) B. velezensis isolate B-27+B. cereus isolate RC76+compost, (D) compost + Trichoderma asperellum and (E) control (compost 1 ton/ha) with 5 replications on glasshouse treatment and 3 replications on field treatment. The results showed that the combination of B. velezensis in compost effectively reduced the incidence of twisted disease, the number of Fusarium spp. colonies, and the number of infected bulbs by Fusarium sp. Besides, the combination of compost with microbial agents showed better results than compost single treatment.

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.

First Report of Neck Rot Caused by Botrytis aclada on Onion (Allium cepa L.) in Gansu Province, China.

Onion (Allium cepa L.) is one of the most important cultivated vegetable crops in many countries, including China (Hanci 2018; Steentjes et al. 2021). Gansu, in the northwest region of China, is a major area of onion production (Zhang et al. 2022). In October 2021, typical symptoms of neck rot were observed on stored onion bulbs (cv. Honghe) in Jiuquan, Gansu Province, China. Further surveys of 20 bags of onion bulbs randomly selected in a storage facility with bulbs harvested from 73 ha indicated that approximately 5% of the bulbs had typical neck rot symptoms. The neck of infected bulbs developed a water-soaked decay, with softened and discolored inner scales with white to gray mycelium in the neck. Infection usually began in the neck and sometimes spread through the entire bulb. In severely affected bulbs, the fleshy scales were span style="font-family:'Times New Roman'">decayed and the bulbs had shrunk, with black sclerotia between the rotting scales in the neck and shoulders of the bulb. Small pieces cut from the margins of lesions were surface-disinfested with 75% ethanol for 10 s, and 1% NaClO for 1 min, rinsed three times in sterile distilled water, dried on sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 20 ± 1℃ for 5 days in the dark. Ten pure cultures were obtained by single-spore isolation (one from each of 10 bulbs). All 10 isolates produced colonies that initially were white, and became gray to dark in color with gray mycelium that was covered with abundant conidia resembling Botrytis species when cultured on PDA at 20 ± 1℃ for 10 days in the dark. Conidia (n = 100 per isolate) were one-celled, ellipsoid or ovoid, beige to dark brown, and 5.6 to 14.8 × 3.9 to 8.5 μm. Sclerotia were not produced by any of the isolates on PDA after incubation at 20 ± 1°C for 30 days. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, heat shock protein 60 (HSP60) gene, and DNA-dependent RNA polymerase II core subunit (RPB2) gene of two representative isolates, JQ21AC03 and JQ21AC09, were amplified and sequenced with primers ITS1/ITS4, G3PDH-F/G3PDH-R, HSP60-F/HSP60-R, and RPB2-F/RPB2-R, respectively (Staats et al. 2005), and deposited in GenBank (ITS: OP604277 and OP604283; G3PDH: OP627512 and OP627515; HSP60: OP627513 and OP627516; and RPB2: OP627514 and OP627517). BLASTn analysis of the resulting sequences showed 98 to 100% similarity with those of Botrytis aclada. A maximum likelihood phylogenetic tree generated by combining the sequenced loci using MEGA11.0, clustered isolates JQ21AC03 and JQ21AC09 with B. aclada with 99% bootstrap support. Based on these results, isolates JQ21AC03 and JQ21AC09 were identified as B. aclada. Pathogenicity of the two isolates on the onion cv. Honghe was confirmed by inoculating 30 heathy onion bulbs per isolate with 100 μL of conidial suspension (1×105 conidia/ml), while control bulbs were inoculated with 100 μL sterile distilled water. All 30 bulbs inoculated with each isolates developed neck rot symptoms after 30 days incubation at 15 ± 1ºC in the dark, with symptoms identical to those observed in the original storage facility, whereas control bulbs remained symptomless. The pathogen was re-isolated from the symptomatic tissue of inoculated bulbs, fulfilling Koch's postulates. hang et al (2008) documented this pathogen causing bulb rot of onions in Wuhan, Hubei Province, 1,700 km from Gansu. To the best of our knowledge, this is the first report of B. aclada causing neck rot on onion bulbs in storage in Gansu Province, which is the main onion production region in China. Considering that onion is the main source of income for growers in Gansu, further studies will be required to understand the epidemiology of this disease and foster appropriate disease management measures to avert disease outbreaks in the future.

Nanopore Metagenomics Sequencing for Rapid Diagnosis and Characterization of Lily Viruses.

Lilies (Lilium spp.) are one of the most important ornamental flower crops grown in Korea. Most viral diseases in lilies are transmitted by infected bulbs, which cause serious economic losses due to reduced yields. Various diagnostic techniques and high-throughput sequencing methods have been used to detect lily viruses. According to Oxford Nanopore Technologies (ONT), MinION is a compact and portable sequencing device. In this study, three plant viruses, lily mottle, lily symptomless, and plantago asiatica mosaic virus, were detected in lily samples using the ONT platform. As a result of genome assembly of reads obtained through ONT, 100% coverage and 90.3–93.4% identity were obtained. Thus, we show that the ONT platform is a promising tool for the diagnosis and characterization of viruses that infect crops.

Preliminary Studies on Detection of Fusarium Basal Rot Infection in Onions and Shallots Using Electronic Nose.

The evaluation of crop health status and early disease detection are critical for implementing a fast response to a pathogen attack, managing crop infection, and minimizing the risk of disease spreading. Fusarium oxysporum f. sp. cepae, which causes fusarium basal rot disease, is considered one of the most harmful pathogens of onion and accounts for considerable crop losses annually. In this work, the capability of the PEN 3 electronic nose system to detect onion and shallot bulbs infected with F. oxysporum f. sp. cepae, to track the progression of fungal infection, and to discriminate between the varying proportions of infected onion bulbs was evaluated. To the best of our knowledge, this is a first report on successful application of an electronic nose to detect fungal infections in post-harvest onion and shallot bulbs. Sensor array responses combined with PCA provided a clear discrimination between non-infected and infected onion and shallot bulbs as well as differentiation between samples with varying proportions of infected bulbs. Classification models based on LDA, SVM, and k-NN algorithms successfully differentiate among various rates of infected bulbs in the samples with accuracy up to 96.9%. Therefore, the electronic nose was proved to be a potentially useful tool for rapid, non-destructive monitoring of the post-harvest crops.

Plantago asiatica mosaic virus: An emerging plant virus causing necrosis in lilies and a new model RNA virus for molecular research.

Taxonomy Plantago asiatica mosaic virus belongs to the genus Potexvirus in the family Alphaflexiviridae of the order Tymovirales.Virion and genome propertiesPlantago asiatica mosaic virus (PlAMV) has flexuous virions of approximately 490–530 nm in length and 10–15 nm in width. The genome of PlAMV consists of a single‐stranded, positive‐sense RNA of approximately 6.13 kb. It contains five open reading frames (ORFs 1–5), encoding a putative viral polymerase (RdRp), movement proteins (triple gene block proteins, TGBp1‐3), and coat protein (CP), respectively.Host rangePlAMV has an exceptionally wide host range and has been isolated from various wild plants, including Plantago asiatica, Nandina domestica, Rehmannia glutinosa, and other weed plants. Experimentally PlAMV can infect many plant species including Nicotiana benthamiana and Arabidopsis thaliana. It also infects ornamental lilies and frequently causes severe necrotic symptoms. However, host range varies depending on isolates, which show significant biological diversity within the species.Genome diversityPlAMV can be separated into five clades based on phylogenetic analyses; nucleotide identities are significantly low between isolates in the different clades.TransmissionPlAMV is not reported to be transmitted by biological vectors. Virions of PlAMV are quite stable and it can be transmitted efficiently by mechanical contact.Disease symptomsPlAMV causes red‐rusted systemic necrosis in ornamental lilies, but it shows much weaker, if any, symptoms in wild plants such as P. asiatica.ControlControl of the disease caused by PlAMV is based mainly on rapid diagnosis and elimination of the infected bulbs or plants.

Sensitivity of bulb tissue section for detection of Fusarium causes Moler disease of shallot

Abstract Moler caused by Fusarium oxysporum f.sp.cepae is a seed-borne disease, so the disease intensity in the field is depend on the amount of initial inoculum in the infected bulbs as seeds. Therefore, the detection of seed-borne pathogens is important to determine the potential of their transmission. The aim of this research was to evaluate the sensitivity of the bulb tissue of shallot based on the pathogen Infection Index by incubation method on agar media to detect the presence of F. oxysporum f.sp. cepae. The experiment was conducted using a completely randomized design with five replications and three varieties of shallots (Thailand, Bima, and Bauji). Each treatment unit was placed in a Petri’s dish with a diameter of 20 cm, containing 40 mL PDA on which 16 pieces of the tip, middle, and base of the bulb tissues were placed, which were incubated for 7 days. Pathogen Infection Index is a parameter that was observed macroscopically and microscopically. The results showed that the base of the bulbs tissue section of the three varieties tested showed higher sensitivity than the other part to detect the presence of F. oxysporum f.sp. cepae in shallot. It indicates that the basal bulb is the major infection side of the pathogen.

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris.

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen, Rhizoctonia tuliparum (Rtul). Sclerotia present in infected bulbs, as well as overwintering sclerotia in soil and field debris, are the primary sources of infection. A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels, is needed for the management of disease caused by this pathogen. We designed a unique set of primers targeting the ITS2 region of the Rtul genome and developed a highly sensitive quantitative PCR (qPCR)-based method for Rtul identification using these primers, where the threshold of detection was approximately 1 fg Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with those grown in the lab, and more sensitive with natural-light than natural-dark sclerotia. Also, the detection method was more sensitive when sclerotia were extracted from soil than from bulb tissue. The qPCR method was highly specific, as no PCR amplification was detected when genomic DNA from 62 non-Rtul Rhizoctonia isolates from a wide range of anastomosis groups were tested. To understand the evolutionary relationships and genomic diversity of Rtul, we performed phylogenetics of the ITS1-5.8S-ITS2 region and ITS2-molecular morphometric characterization (MMC) of Rtul isolates. The three Rtul isolates whose ITS sequences were available in GenBank formed a distinct phylogenetic clade with Ceratobasidium anceps as the nearest relative. Furthermore, MMC analysis revealed genetic divergence among these three Rtul isolates.

The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like Arthrobotrys oligospora or Duddingtonia flagrans produce adhesive trapping networks to catch and immobilize nematodes. After penetration of the cuticle, hyphae grow and develop inside the worm and secrete large amounts of hydrolytic enzymes for digestion. In many microbial pathogenic interactions small-secreted proteins (SSPs) are used to manipulate the host. The genome of D. flagrans encodes more than 100 of such putative SSPs one of which is the cysteine-rich protein CyrA. We have chosen this gene for further analysis because it is only found in NTF and appeared to be upregulated during the interaction. We show that the cyrA gene was transcriptionally induced in trap cells, and the protein accumulated at the inner rim of the hyphal ring before Caenorhabditis elegans capture. After worm penetration, the protein appeared at the fungal infection bulb, where it is likely to be secreted with the help of the exocyst complex. A cyrA-deletion strain was less virulent, and the time from worm capture to paralysis was extended. Heterologous expression of CyrA in C. elegans reduced its lifespan. CyrA accumulated in C. elegans in coelomocytes where the protein possibly is inactivated. This is the first example that SSPs may be important in predatory microbial interactions.

First report of soft rot of onion caused by Pectobacterium wasabiae in Japan.

Onion (Allium cepa L.) is one of the important vegetables in Japan. In the summer of 2019, onions with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. Diseased onion showed chlorosis, maceration of leaves, and rotted bulbs. We sampled some diseased onions and isolated three independent isolations (NAONI191, NAONI192 and NAONI193) from infected bulbs on LB medium. These strains were identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All the strains exhibited pectolytic activity on potato slices. DNA from these strains yielded a single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR. However, DNA from these strains did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. The sequence analysis of 16S rDNA (LC597917- LC597919) showed more than 98% identities to P. wasabiae strains (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597925- LC597927), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597972-LC597974), isocitrate dehydrogenase (icdA, LC597998- LC597998LC598000), malate dehydrogenase(mdh, LC598024- LC598026), mannitol-1-phosphate dehydrogenase (mtlD, LC598050- LC598052), glucose-6-phosphate isomerase (pgi, LC598076- LC598078) and gamma-glutamyl phospate reductase (proA, LC598099- LC598101)), and all clustered into a clade containing other confirmed strains of P. wasabiae. As a result, these three strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these three strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against onion (cv. 'Hayate') was confirmed by the field experiments with 5 weeks growth plants sprayed with bacterial suspension (1×107cfu/ml) resulting in soft rot on the plants about four weeks after inoculation, whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of onion in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of onion in Japan.

Identification of specific odour compounds from garlic cloves infected with the potato tuber nematode, Ditylenchus destructor, using gas chromatography-olfactometry

Summary The potato tuber or potato rot nematode, Ditylenchus destructor, causes severe damage to garlic (Allium sativum) produced in Japan. Although consumption of a nematode-infected garlic bulb is not harmful to human health, it causes consumer dissatisfaction because the infected bulb deteriorates quickly. In addition, nematode-contaminated garlic cloves are inadvertently used as seed garlic for next season, which then leads to nematode-infested fields and increases yield loss. However, infected garlic bulbs look the same as healthy ones, and hence have been unknowingly distributed and sold. A method is needed to discriminate infected garlic bulbs from non-infected garlic bulbs before distribution in the market, but to date no suitable technique has been developed. The objective of this study was to identify specific odour-active compounds associated with nematode-infected garlic through the use of analytical chemistry. Garlic cloves were infected with cultured nematodes under controlled conditions, and the volatile odour-active compounds were analysed by an ‘odour identification system’ which included gas chromatography-olfactometry (GC-O), GC-fractionation (GC-F), GC-mass spectrometry (GC-MS), and odour/aroma-specific database. Two specific odour-active compounds associated with nematode-infected garlic cloves were identified as allyl methyl disulfide and eugenol, and two others were tentatively identified as (E)-1-allyl-2-(prop-1-en-1-yl)disulfane and allyl methyl trisulfide. These specific odour-active compounds may be useful as indicators to detect nematode-infected garlic bulbs in a non-destructive manner.

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.

Monitoring the incidence of dry rot caused by Fusarium proliferatum in garlic at harvest and during storage

Abstract Dry rot is an emerging postharvest disease of garlic (Allium sativum) attributed to Fusarium proliferatum, which has caused huge economic losses in the past few years. In this study, we aimed to detect the presence of F. proliferatum on garlic bulbs postharvest during prolonged storage, and to identify other fungal species associated with garlic dry rot. We also quantified the level of fumonisins in symptomatic and asymptomatic cloves. A total of 100 plants were sampled from three production seasons at six farms located in Northern Italy at three time points (at harvest, processing, and 6 months post-storage at –4 °C). The Fusarium–garlic pathosystem was split into two parts: basal plate/root and bulb. F. proliferatum was the dominant fungus in infected bulbs and was confirmed as the causal agent of dry rot in garlic postharvest (mean incidence: 35.4 %). F. oxysporum co-occurred with F. proliferatum but caused disease only in the basal plate/root. Dry rot incidence slightly increased during cold storage (from 14.6 % at processing to 18.4 % at 6-month storage). Although F. proliferatum incidence was stable during cold storage, fumonisins were produced. Symptomatic cloves were more contaminated than asymptomatic cloves, both by the fungus (mean incidence 39 % vs. 25.3 %) and the toxin (287.0 vs. 24.4 μg kg−1). These results suggest that cold storage delays the progression of dry rot, but the risk of health issues related to fumonisins and the occurrence of infection in asymptomatic cloves should be seriously considered.