Bulb Rot Research Articles

The Potential of Rhizophagus intraradices and Trichoderma asperellum to Induce Shallot Resistance against Twisted Disease

Twisted disease caused by Fusarium spp. is one of the primary diseases on shallots with potency to cause enormous losses by causing stunting and bulb rot. One alternative disease control is the induction of plant resistance since the seed stage. The aim of this study was to determine the content of salicylic acid, jasmonic acid, and phenolic compounds of shallot bulb seeds coated with biological control agents as a resistance response to twisted disease. The shallot cultivar used was Crok kuning. The treatments used in this study were the type of biological control agents, including Rhizophagus intraradices and Trichoderma asperellum, combined with and seed coating application time (one month before planting and simultaneously at planting). Biological control agents in the form of powder formulation applied as seed coating material and seeds were stored for one month before planting. The results showed that application of biological agents delayed the occurrence of the twisted disease symptoms. The salicylic acid content from plant treated with R. intraradices at the time of planting was slightly higher than the control. The jasmonic acid content in T. asperellum plants treated at planting was higher than then control. Total phenolic content from plants treated with T. asperellum at planting time was higher than the control. In general, application of biological control agent as seed coat did not result in significant increase in salicylic acid, jasmonic acid nor the phenolic compounds, compared to the pathogen infected control.

The First Report of Juglans regia Leaf Spot Caused by Fusarium proliferatum in China.

Juglans regia is planted widely in China because of its significant economic value as a woody oil species. In August 2019, leaf spots were discovered in a walnut orchard in Tang County, Baoding City, Hebei Province. In the early stages of the disease, irregular brown spots appeared on the edges of leaves, causing them to shrivel; in a later stage, the disease spread inward along the edges to the whole leaves, which then withered. The diseased leaves were collected from the walnut orchard. Select 3-4 mm2 of diseased and healthy junction for each diseased leaf, and the surfaces were sterilized with 75% ethanol for 30 s and 0.1% HgCl2. The leaves were incubated on potato dextrose agar (PDA) at 28 ℃ in darkness, each diseased leaf was repeated 10 times, and purified after 6 days. The diameters of the suspected Fusarium isolates reached 8.3 cm, and the aerial mycelia were white to cream and wool-like, later turning violet and dark purple with age. On PDA, the macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). For molecular analysis, DNA was extracted from the mycelium and used the specific primers to amplify the translation elongation factor EF-1α, and RNA polymerase II largest-subunit (RPB2) further. The amplicons were sequenced and deposited in GenBank under accession numbers MZ891582, and MZ891583. BLAST analysis revealed that all these sequences showed more than 99% sequence identification with F. proliferatum. We used Fusarium MLST for the identification of this strain and the results showed that EF-1α and RPB2 genes have more than 99% similarity with the database F. proliferatum data, with 100% sequence duplication(Deposited of EF-1α in GenBank under accession numbers: MH582346, and MH582347; Deposited of RPB2 in GenBank under accession numbers: MH582193, MH582151, MH582196, and MH582172). We created a multigenic tree using Fusarium MLST. Based on the results of the morphology tests and the molecular sequences, the isolates from the diseased leaves were identified as F. proliferatum. To confirm the pathogenicity, healthy walnut leaves of tree planted in pot were inoculated using 50 µL of conidia suspension (1.0×107 CFU) from a pure clone, and those inoculated using only distilled water were taken as controls. All treatments were repeated three times and placed under 28°C, RH 100% for 5 days. The inoculated plants showed the same symptoms as those observed in the original diseased plants, while the control plants were asymptomatic. The same fungus was isolated again from the inoculated leaves of tree planted in pot. The re-isolated pathogen from diseased plants was identical to the purified strain used for inoculation, confirmed Koch's postulates. According to the above results, F. proliferatum was identified as the pathogen behind the Juglans regia leaf spots, and it is clearly different from other pathogens, such as Colletotrichum gloeosporioides. It is reported that F. proliferatum can lead to necrotic leaf lesions and the bulb rot of stored onions (Beck et al., 2020), as well as light brown discoloration and dryness on palm leaves and branches (Alananbeh et al., 2021). At the same time, F. proliferatum has been found to have the highest colonization ability on jujube leaves and is becoming a serious pathogen on jujube (Saleh et al., 2016). To our knowledge, this is the first report of walnut leaf spots caused by F. proliferatum in China.

Performance of a semi-glossy onion hybrid in certified organic onion fields infested with Thrips tabaci and bulb-rot causing bacteria

More than one-third of the certified organic farms that produce dry bulb onions in the USA are in the Great Lakes region, while onion thrips, Thrips tabaci Lindeman, and bulb-rot causing bacteria are important onion production constraints. Onion cultivars with semi-glossy foliage have been reported to support fewer thrips and suffer less thrips damage and therefore may provide a non-chemical option for thrips management. However, inconsistent results regarding efficacy of semi-glossy onions to reduce thrips densities have been reported in field studies. Bacteria causing bulb rot can infect onion plants through wounds in leaves, but the relationship between thrips damage and bacterial bulb rot disease needs further investigation. In this study, the performance of a semi-glossy onion hybrid, ‘USDA Maia’, was compared with the commonly grown waxy, thrips-susceptible cultivar ‘Bradley’ on certified organic farms in New York and Wisconsin from 2019 to 2021. Results indicated that thrips abundance was not different between ‘USDA Maia’ and ‘Bradley’. Bacterial bulb rot incidence in ‘USDA Maia’ was either similar or lower than the incidence in ‘Bradley’ in four of six trials, but higher in the other two. Marketable bulb yield in ‘USDA Maia’ was similar to yield in ‘Bradley’ in four of seven trials, but lower in the other three. Positive relationships between thrips density and bacterial bulb rot incidence occurred in one of three trials for both onion cultivars. Similarly, negative relationships between thrips density and marketable bulb yield only occurred in one of three trials, regardless of onion cultivar. ‘USDA Maia’ did not reduce thrips infestations as anticipated, but it was similar in susceptibility to bacterial bulb rot disease and produced similar large bulb yields as ‘Bradley’. While high thrips abundance can potentially increase incidence of bacterial bulb rot disease, wet growing conditions during bulbing was more important.

Determination of Resistance Levels of Some Onion Cultivars or Inbreed Lines with Fusarium Testing at Seedling Stage

This study was carried out to determine the resistance levels of some onion genotypes in Yalova onion gene pool by Fusarium testing during seedling stage. The isolate used in the study was Fusarium oxysporum f. sp. cepae, which causes damping off during the seedling stage and later basal rot in onion bulbs. The variance analysis for the onion seedling test and the mean differences against control were analyzed by using General Linear Model of the Tukey test. The germination rate of control seeds varied between 72% to 98%, while the germination rate of inoculated seeds varied between 39% to 93%. Texas Early Grano 502 showed the highest level of resistance with a survival rate of 83.8%, and resistance levels of 19Y07 and 19Y142 genotypes were higher than other genotypes. Resistance levels of 19Y51, 19Y15 and 19Y73 genotypes were lower than other genotypes. Akgün 12 showed moderate resistance with a survival rate of 59.6%. Determining the resistance levels of these onion genotypes during the seedling stage may be a preliminary step towards further studies.

Phylogeny and Identification of Pantoea Species Associated with Bulb Rot and Bacterial Leaf Blight of Onion Crops in Uruguay.

Onion is among the most consumed vegetables in Uruguay, grown in the northwestern and southern regions of the country. The onion supply presents interannual variations associated with significant postharvest losses, mainly caused by bacterial rots. Besides bulb rotting, onion leaf lesions as well as infections on seed-stalks during seed production may be devastating for some varieties under conducive conditions. This research aimed to identify the causal agents of bulb rots and leaf blight of onion crops in Uruguay. Symptomatic bulbs, seeds-stalks, and leaves were collected from commercial fields from 2015 to 2020. Bacterial colonies were isolated and identified at genera level using physiological tests and 16S rRNA gene sequence analysis. A collection of 59 Pantoea spp. isolates was obtained (11 from bulbs and 48 from leaves and seeds-stalks). Multilocus sequence analysis using four housekeeping genes (rpoB, gyrB, leuS, and fusA) allowed the assignment of the isolates to five Pantoea species: P. ananatis, P.agglomerans, P. allii, P. eucalypti, and P. vagans. The last two species were not previously reported as onion pathogens elsewhere. The ability to cause disease symptoms was tested by leaf inoculation and red onion scale assays. P. ananatis isolates showed the highest aggressiveness in both assays. Specific isolates from P. allii (MAI 6022), P. eucalypti (MAI 6036), P. vagans (MAI 6050), and Pantoea sp. (MAI 6049) ranked second in aggressiveness on onion leaves, whereas only three isolates belonging to P. eucalypti (MAI 6036 and MAI 6058) and P. agglomerans (MAI 6045) exhibited the same scale-clearing phenotype as P. ananatis. Leaf inoculation assays were also performed on a set of eight onion cultivars and breeding lines. Overall, P. ananatis MAI 6032 showed the highest aggressiveness in all tested cultivars, followed by P. eucalypti MAI 6036. The presence of new reported bacterial species leads to complex disease management and highlights the need for further studies on virulence factors and the epidemiology of these pathogens.

Efficacy of chemical and biological spray seed treatments in preventing garlic dry rot

Garlic dry rot caused by Fusarium proliferatum is an emerging postharvest disease that has resulted in severe economic losses, necessitating design and implementation of efficient disease control strategies. Sanitation of planting cloves is critical for preventing garlic dry rot. This study evaluated the efficacy of commercial chemicals and biocontrol agents, applied at planting as spray treatments, for reducing disease severity and the occurrence of Fusarium spp. in garlic, from the field stage then through 9 months of postharvest storage. Tebuconazole was the most effective for reducing disease severity, giving 26.5% reduction of basal plate rots and 44% reduction of bulb rots, and 33.4% reduction in visible symptoms on cloves relative to the untreated controls. Comparable results were obtained by applying B. subtilis and S. griseoviridis. However, none of the active ingredients tested in this study reduced the incidence of F. oxysporum and F. proliferatum on basal plates, although tebuconazole reduced the postharvest incidence of F. proliferatum on cloves by nearly 50%. Incidence of F. proliferatum increased by 37% in bulbs transferred from storage to room temperature (25°C) for 15 days, simulating storage in consumers’ homes. These results demonstrate that spray seed clove treatments have inhibitory effects on postharvest garlic dry rot, although further research is required to determine the persistence of these treatments during prolonged storage, especially without low temperatures.

First Report of Onion (Allium cepa) Bulb Rot Caused by Pantoea agglomerans in China.

Onion (Allium cepa L.) is one of the most cultivated vegetables throughout the world. With an average annual production quantity of 18 million kg in recent 21 years, China is the world's biggest onion producer (Hanci F., 2018). Among them, onion is mainly cultivated in the provinces of Gansu, Shandong, Yunnan, Jiangsu, Zhejiang and Henan. A survey in Gansu province in last several years showed that the incidence of onion bulb rot was 30%-80%. In April 2020, bulbs displayed water-soaked, and then rot symptoms observed during storage in Lanzhou City, Gansu Province, China. The initial symptoms of bulb rot disease were yellowish brown, and produced an abundant exudate in the inner bulb scales when cut. Gradually, symptomatic bulbs became soft, watery and decayed. In severe infections, the onions showed total rot of the bulb. Therefore, we sampled some diseased onions and isolated pathogenic bacteria from the junction of lesion along with healthy parts on Luria-Bertani (LB) medium. Three representative single colonies were obtained on LB medium, and the culture characteristics were raised elevation, mucoid texture, round, and smooth with entire margin, the brown at the beginning and turned yellow later, and scanning electron microscopy (SEM) observations showed that these isolates were short rod-shaped (Fig. 1A). The physiological and biochemical determination revealed that the isolates were positive for yellow pigment, v-p test, growth at 37 ℃, nitrate reduced, catalase, glucose, sucrose, D (-)-salicin, starch hydrolysis, motility, pellicle. On the contrary, they were negative for indole production, methyl red, lactose, gelatin liquefaction, glycerol, gram staining (Gavini et al., 1989; Nabrdalik et al., 2018). Based on these morphological, physiological and biochemical characteristics, three isolates were initially identified as Pantoea agglomerans (Guo et al., 2020). A representative isolate L1 was selected to extract DNA, and the conserved sequences of the pathogen gene were sequenced according to 23S ribosomal RNA (23S rRNA), DNA gyrase subunit B (gyrB), elongation factor G (fusA) (She et al., 2021) housekeeping gene. The sequence alignment of the 23S rRNA gene (P. agglomerans, MZ314289, 930bp) showed that the homology between the strain L1 and P. agglomerans (CP016889) with similarity of 99.54%, and based on the sequence alignment of gyrB (P. agglomerans, MZ337547, 1189bp) and fusA (P. agglomerans, MZ350961, 1037bp) genes, the similarity with P. agglomerans (FJ617386 and MG845872) was 100%. A phylogenetic analysis based on the 23S, gyrB, fusA housekeeping gene sequences was performed by using the neighbor-joining methods in MEGA 7.0 under the p-distance (Kumar et al, 2016), which included P. agglomerans strains AR1a, TH81, L15, ASB05, P. eucalypti strain LMG 24197, P. dispersa strains BJQ0007 and DSM 32899, P. ananatis strains LMG 20103 and AJ13355, P. vagans strains C9-1, LMG24199 and PV989. The phylogenetic distribution generated five primary phylogroups, and strain L1 formed a clade with the other four P. agglomerans strains (Fig. 2). Thus, the strain L1 was identified as P. agglomerans. To satisfy Koch's postulates, ten onions were divided into two groups, five in each group, and needle punctured wound on the surface of each onion. In the experimental group, 400 μL bacterial suspensions were injected with sterile syringe, and the other five onions were injected with the same amount of sterile distilled water as the negative conrol. Inoculated onions were incubated in the greenhouse incubator (28 ℃, humidity > 80%). After 4 days of incubation, all onions inoculated with strain L1 appeared water-soaked, rot symptoms, and no symptoms were observed in the negative control (Fig. 1B). Subsequently, pathogens were re-isolated from inoculated bulbs and identified as P. agglomerans according to molecular identification described above. To the best of our knowledge, this is the first report that the bulb rot disease of stored onion caused by P. agglomerans in China.

Identification of Pseudomonas viridiflava, causal agent of onion (Allium cepa L.) bulb rot

Objective: To phenotypically and molecularly characterize and identify the causal agent of onion (Allium cepaL.) bulb rot in Morelos, Mexico.Methodology: Fluorescent bacteria from onion bulb tissue with symptoms of rot were isolated; the LOPATtest was used to describe them and subsequently they were identified by partial 16S rRNA gene amplification.Pathogenicity in vegetables and plants was evaluated injecting a suspension with 108 CFU mL-1of the pathogen.Results: Phenotypic characterization and 16S rRNA nucleotide sequencing showed 100% identity withPseudomonas viridiflava as the causal agent of onion bulb rot. The pathogen caused infection in broccoli (Brassica oleracea L.), spring onion (Allium fistulosum L.), purple onion (Allium cepa L.), cauliflower (Brassica oleracea var. botrytis L.), leek (Allium porrum L.), and carrot (Daucus carota L.), as well as in plant species such as jalapeño pepper (Capsicum annuum var. annuum L.), bean (Phaseolus vulgaris L.), and tomato (Solanum lycopersicum L.).Implications: This information is important for agriculture in Mexico. Pseudomonas viridiflava is a bacterialpathogen with high potential to infect new hosts. This is the first report of P. viridiflava causing onion rot in Mexico.Conclusions: Pseudomonas viridiflava is the causal agent of onion bulb r

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.

First report of Fusarium solani associated with the bulb rot of Tulip (Tulipa spp) in India.

Tulip is an ornamental bulbous flowering crop belonging to the Genus Tulipa and family Liliaceae. It is the first ranking bulbous ornamental plant in the world (Nayeem and Qayoom 2015). They are often the first flowers to witness the bloom in the spring. Kashmir valley is located in northern Himalayas in northwestern region of Indian subcontinent. It is the most alluring and fascinating place all over India and the home of famous "Indhra Gandhi Memorial Tulip garden", the largest tulip garden in the entire Asia. However there are number of constraints in tulip cultivation among which bulb rot occupy a prominent place (Piwoni 2000). Bulb rot is posing problem to all the tulip growers throughout the world (De Hertogh et al. 1983). Rot symptoms were observed on tulip bulbs in field as well as in storage conditions (20-22◦C temperature with a relative humidity of 65%) in the summers of 2018 and 2019 in Shalimar fields of Kashmir. The main disease symptoms are yellow sunken spots on bulbs, purple-yellow coloration of leaves. Causal agent was isolated using tissue bit technique (Pathak 1972) on potato dextrose agar plates which where incubated at 24±2◦C . Single spore technique was used to obtain the pure isolate (Johnston and Booth 1983). The isolate covered the full plate (90mm) in ten days. The colony was dull whitish in color, flat and smooth with concentric ring formation in the culture plate with inner ring having a creamy exudation. The mycelium was septate, branched and hyaline in color and measured 3.50-5.20 µm in width with an average of 4.4 µm. Micro-conidia were hyaline, cylindrical to oval, 0-1 septa and measured 7.50-11.00×2.80-3.75 µm in size. Macro conidia were hyaline with 3-4 septa, fusiform, moderately curved which measured 21.15- 32.00×3.80-4.75 µm in size with an average of 28.50±0.21× 4.30±0.2 µm. On the basis of these morphological and cultural characteristics of the fungus, it was identified as Fusarium solani (Mar.) Sacc.,. To confirm the identity the PCR amplification was carried out for two genes Internal Transcribed Spacer (ITS 1, ITS 4)and Translation Elongation factor1-alpha gene (tef1- alpha) (O'Donnell et al. 1998; White et al. 1990). BLAST analysis of the sequence obtained for both the genes showed 99% homology with F. solani sequences in GenBank and Fusarium -ID databases. The sequences were deposited in the GenBank (Accession No MN611433, MW995477). Pathogenicity test was conducted on variety orange emperor both in laboratory and polyhouse. Bulbs were divided into three sets, (three bulbs per set) one set was given injury and dipped in conidial suspension (106 conidia/ml) for 30 min, another set was kept uninjured and dipped in spore suspension of same concentration, the third set was served as control and dipped in sterilized distilled water. All the respective sets were incubated in a moist chamber maintained at a temperature of 22 ◦C to observe symptoms. The injured ones showed symptoms after 7-8 days of inoculation, whereas the uninjured bulbs showed symptoms after 11-12 days. No symptoms were observed in controlled set. A pot experiment was also conducted to carry the pathogenicity tests. Bulbs were injured with the help of sterile needle and were dipped in conidial suspension (106 conidia/ml) for 30 min (Pastrana et al. 2014). The bulbs kept for control were dipped in sterilized distilled water. Bulbs were then planted in pots maintained at 18◦C. The above ground parts of the inoculated bulbs showed symptoms like stunted growth which gradually turned yellow and did not produced flowers. The bulbs after harvesting were rotten .No symptoms were observed in controlled plants. To fulfill the Koch's postulates the fungal pathogen was re-isolated which was identified as F. solani. The pathogen is reported to cause disease in other crops (Gupta et al. 2012) but to our knowledge and on the basis of literature, this is the first report of F. solani causing bulb rot of tulip in India.

Comparative transcriptome and proteome analysis of lily clones inoculated with <i>Fusarium oxysporum</i> f. sp. <i>lilii</i>

Basal bulb rot is the major factor restricting the production of lily, caused by <italic>Fusarium oxysporum</italic> f. sp. <italic>lilii</italic>. To systematically analyze the transcriptomic and proteomic responses of lily clones to <italic>F. oxysporum</italic>, we constructed six RNA-seq libraries and four iTRAQ proteomic libraries using lily resistant and susceptible clones sampled at 0, 24 and 48 h post-inoculation (hpi) with <italic>F. oxysporum</italic> respectively. 137,715 unigenes were generated, of which 7,667 were differentially expressed. 1,679 and 4,051 differentially expressed genes (DEGs) between resistant and susceptible clones were isolated from samples collected at 0 and 48 hpi. 433 and 155 DEGs were identified in resistant clones sampled at 24 and 48 hpi separately while 550 and 799 DEGs were isolated in the susceptible clones sampled at 24 and 48hpi respectively. The results of iTRAQ analysis revealed 7,482 proteins in resistant and susceptible clones. Data analysis of transcriptome and proteome indicated that 5,735 proteins corresponded to mRNAs. 316 and 1,052 DEGs had corresponding DEPs. At 48 hpi, the resistant clones showed 155 DEGs and 108 corresponding DEPs, while the susceptible clones showed 799 DEGs and 316 corresponding DEPs. In general, these results enhance comprehend of the defense response of lily resistant clones to <italic>F. oxysporum</italic> infection and provide valuable sequence data for studying the resistant mechanism.

First Report of Fusarium proliferatum Causing Garlic clove Rot in Russian Federation.

Garlic (Allium sativum L.) is a widely consumed bulbous crop both worldwide and in Russia. About 200,000 tons of garlic is produced in Russia annually (https://rosstat.gov.ru/). Significant pre- and post-harvest losses of garlic regularly occur due to Fusarium sp. (Taylor et al., 2013). Since September 2018, rotting has been observed in Russia during garlic bulb storage (data of the Federal Scientific Vegetable Center, FSVC, Moscow Region). The outer bulb surface looked healthy, but underneath the integumentary scales, the cloves had light brown and brown spots. When grown, diseased plants were characterized by root and bulb disruption and leaf drying; for some cultivars, up to 100% of plants died. In January 2020, cv. Strelets and Dubkovsky bulbs, collected in July 2019, with rot symptoms, were taken from the FSVC storage. Necrotic clove tissue fragments (0.2-0.5 cm) were cut, sanitized with 70% ethanol for 3 min, rinsed with sterile water, and incubated on potato dextrose agar (PDA) with 1 mg/ml ampicillin at 22°C in the dark. Four single-spore cultures were obtained from four diseased bulbs. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. All isolates (Dubkovsky, Dubkovsky 2, Strelets, and Strelets 2) produced numerous oval unicellular microconidia without septa, 4.1 to 11.6 × 1.3 to 3.4 µm (n = 50) and very few macroconidia with 3-4 septa (21 to 26 × 3 to 4 µm (n = 30)), narrowed at both ends. The cultural and conidial characteristics of the isolates corresponded to Fusarium species (Leslie and Summerell 2006). To determine the species, DNA was extracted from four isolates, and the internal transcribed spacer (ITS), and genes of translation elongation factor 1α (EF1α) and subunits 1 and 2 of DNA-directed RNA polymerase II (RPB1 and RPB2) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 1998a), RPB1-F5/RPB1-R8 (O'Donnell et al. 2010) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained sequences were identical for all four isolates. The isolate Strelets sequences were deposited in NCBI GenBank (MW149129 (ITS), MW161161 (EF1α), MW413302 (RPB1) and MW413303 (RPB2)); their analysis in MLST (http://fusarium.mycobank.org) showed 98.8-99.8% similarity to F. proliferatum (NRRL 13582, 13598 and others), which is part of the F. fujikuroi complex (O'Donnell et al. 1998b). The test on pathogenicity was performed two times according to (Leyronas et al. 2018). For this, three replicates of 10 cloves (cv. Strelets) were soaked in a conidial suspension (~106 conidia/ml; Strelets isolate) for 24 h. Ten control cloves were soaked in sterile water. The cloves were incubated on Petri dishes (5 cloves on a dish; on filter paper wettened with sterile water) in the dark at 23°C. After 5 days, brown lesions and white mycelium developed on the surface of the treated cloves. The taxonomic status of the fungus isolated from necrotic tissue was determined as F. proliferatum according to the ITS, EF1α, RPB1 and RPB2 analysis. Garlic basal and bulb rot is known to be caused by F. oxysporum f. sp. cepae and F. proliferatum (Snowdon 1990). This study is the first report of F. proliferatum causing rot of garlic bulbs during storage in Russia. F. proliferatum produces a variety of mycotoxins during bulb infestation, and our findings are important for diagnosing a Fusarium disease and the use of garlic crop in culinary and medicine. Funding The reported study was funded by Russian Foundation for Basic Research, project number 20-316-70009.

The first report of bacterial streak and rot of onion caused by Pseudomonas viridiflava in Japan.

In March 2020, a bacterial streak and rot symptom was observed on the onion (Allium cepa L.) leaves in Akita Prefecture of Japan. On the beginning, oval and dark-greenish water-soaked lesions with grayish-white necrotic center, 2-3 mm in diameter, appeared on the middle or the tip of upper leaves. Lesions, frequently surrounded by light yellow halo, expanded along veins and overlapped together. As lesions grew, the center of the lesions turned to light brown necrosis. The basal areas of diseased leaves often rotted, causing the withering of a whole leaf at last. From the water-soaked tissues of young lesions, a bacterium forming cream white colonies and producing fluorescent pigment on King's medium B was consistently isolated, and suggested to be a member of genus Pseudomonas. The isolates were positive for potato soft rot and tobacco hypersensitive reaction, and negative for levan production, oxidase and arginine dihydrolase activity, indicating that they belonged to LOPAT group II, Pseudomonas viridiflava, defined by Lelliott et al. (1966). P. viridiflava is known as a pathogen of bacterial streak and bulb rot of onion in United States and Uruguay, though it has not been reported in Japan. Four isolates were chosen for further examinations. Pathogenicity tests on onion leaves stubbed with selected isolates reproduced similar symptoms as observed in field's samples. After 2 days of inoculation, isolates produced water-soaking around the stabbed holes, developed into grayish-white necrosis on 4 days after inoculation. After 10 days of inoculation, lesions grew to necrotic streaks with light blown center surrounded by water-soaking and light yellow halo. Additionally, on onion scales, isolates caused water-soaked rot, yellow- to brown-colored, in 1-2 days after inoculation. From water-soaked areas of both of leaves and scales, inoculum was reisolated and fulfilled Koch´s postulates. In phenotypic properties, isolates showed consistent results as P. viridiflava strains identified in former studies. They were gram negative and aerobic, positive for hydrolysis of esculin, casein and gelatin, and negative for nitrate reduction and indole production. Regarding the utilization of carbohydrates, positive for 2-keto-gluconate, glucose, fructose, D-galactose, mannose, L(+)arabinose, glycerol, mannitol, sorbitol, myo-inositol, meso-erythritol, meso-tartrate, D(-)-tartrate, gluconate, n-caprate, dl-malate, citrate, L-arginine, L-aspartate, L-glutamate, and negative for D-arabinose, maltose, sucrose, raffinose, adonitol, trehalose, L-tartrate, L-rhamnose, acetate. The 16S rRNA gene sequences (Frank et al 2008) of four isolates (1,377 bp) showed 100% similarity as that of P. viridiflava type strain LMG 2352T (GenBank Accession No. Z76671) in BLASTN search. In phylogenetic analysis using gyrB (910 bp) and rpoD (801 bp) genes (Maeda et al 2006), isolates formed a cluster with P. viridiflava strains deposited in public databases, independent from other closely related Pseudomonas species. Sequences of 16S rRNA (GenBank Accession Nos. LC597475-LC597478), gyrB (LC597479-LC597482) and rpoD (LC597483-LC597486) genes were deposited in DNA Data Bank of Japan. According to these results, the isolates were identified as P. viridiflava (Burkholder 1930) Dowson 1939. This is the first report of the occurrence of bacterial streak and rot of onion caused by P. viridiflava in Japan, causing severe damage on onion growth.

Biocontrol Mechanism of Bacillus subtilis C3 Against Bulb Rot Disease in Fritillaria taipaiensis P.Y.Li.

Bulb rot disease has become one of the main diseases that seriously affects the yield and quality of Fritillaria taipaiensis P.Y.Li (F. taipaiensis). In this study, F. taipaiensis was used as the research object to explore the effect and mechanism of Bacillus subtilis C3 in preventing and curing bulb rot. Through isolation and verification of the pathogenic fungi, we determined for the first time that the pathogenic fungus that causes bulb rot in F. taipaiensis is Fusarium oxysporum. The results of the study showed that B. subtilis C3 inhibits the growth of pathogenic fungi, and the inhibition rate is as high as 60%. In the inhibition mechanism, strain C3 inhibits the conidiogenesis of pathogenic fungi and destroys the cell structure of its hyphae, causing protoplast exudation, chromatin concentration, DNA fragmentation, and ultimately cell death. Among the secondary metabolites of C3, antimicrobial proteins and main active components (paeonol, ethyl palmitate, and oxalic acid) inhibited the growth of F. oxysporum. The molecular weight of the antibacterial protein with the highest inhibition rate was approximately 50 kD. The results of a field experiment on the Taibai Mountain F. taipaiensis planting base showed that after the application of strain C3, the incidence of bulb rot in Fritillaria was reduced by 18.44%, and the ratio of bacteria to fungi in the soil increased to 8.21, which verified the control effect of C3 on Fritillaria bulb rot disease. This study provides a theoretical basis for the use of B. subtilis C3 to prevent and control bulb rot in Fritillaria.

Pathogenicity and Detection of Phytohormone (Gibberellic Acid and Indole Acetic Acid) Produced by Fusarium spp. that Causes Twisted Disease in Shallot

The twisted disease is one of the essential diseases in shallots caused by Fusarium spp. This study aimed to study pathogenicity and identify Fusarium species isolated from shallot plants with twisted symptoms in Nganjuk and Bantul areas. The Fusarium isolates were identified and then tested for pathogenicity levels and the effect of the hormones GA3 and IAA on shallot symptoms. Molecular identification using NF2 and NF4 successfully identified one isolate of Fusarium oxysporum, three isolates of F. acutatum, and three isolates of F. solani. Each of these species produces different symptoms. Pathogenicity test showed that all isolates had disease incidence reaching 100%, except isolates of F. solani1 causing wilt and F. solani3 causing twisted have the lower disease incidence were 77.8% and 77.7%, respectively. The investigation caused twisted shallot related to different symptoms was tested using the Thin Layer Chromatography (TLC) method. The result indicates that all isolates did not find IAA hormone. In contrast, the hormone GA3 was found in F. solani2 and F. solani3 isolates, caused bulb rot and twisted disease, respectively. Detection of IAA, GA3, and other hormones in shallot plants showed different symptoms should be studied further.

Incidence and Etiology of Postharvest Fungal Diseases Associated with Bulb Rot in Garlic (Alllium sativum) in Spain.

In recent years, different postharvest alterations have been detected in garlic. In many cases, the symptoms are not well defined, or the etiology is unknown, which further complicates the selection of bulbs during postharvest handling. To characterize the different symptoms of bulb rot caused by fungi, garlic bulb samples were collected from six Spanish provinces in two consecutive years. Eight different fungal species were identified. The most prevalent postharvest disease was Fusarium dry rot (56.1%), which was associated with six Fusarium species. Fusarium proliferatum was detected in more than 85% of symptomatic cloves, followed by F. oxysporum and F. solani. Pathogenicity tests did not show a significant correlation between virulence and mycotoxin production (fumonisins, beauvericin, and moniliformin) or the mycelial growth rate. Penicillium allii was detected in 12.2% of the samples; it was greatly influenced by the harvest season and garlic cultivar, and three different morphotypes were identified. Stemphylium vesicarium and Embellisia allii were pathogenic to wounded cloves. Some of the isolated fungal species produce highly toxic mycotoxins, which may have a negative impact on human health. This work is the first to determine the quantitative importance, pathogenicity, and virulence of the causative agents of postharvest garlic rot in Spain.

Morphological and molecular identification of Fusarium oxysporum f. sp. narcissi on poet’s daffodil (Narcissus poeticus L.) in Serbia

In autumn 2018, daffodil (narcissus) plants with basal bulb rot were noticed in a plantation in locality Jasika, Serbia. Morphological characterization identified Fusarium oxysporum f. sp. narcissi as the causal agent of bulb rot in daffodil. Three selected isolates were tested for their ability to produce symptoms on daffodil bulbs. Two weeks after artificial inoculation typical symptoms of basal rot developed on inoculated bulbs from which the pathogen was re-isolated fulfilling Koch’s postulates. To further identify the pathogen, a molecular characterization with sequence and phylogenetic analysis of ITS and TEF1 genomic regions was performed. Molecular characterization confirmed the obtained results in morphological identification. The detection of Fusarium oxysporum f. sp. narcissi, a causal agent of basal rot on daffodil in Serbia, is the first report of this pathogen in the country.

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.

Visualization of Three Sclerotiniaceae Species Pathogenic on Onion Reveals Distinct Biology and Infection Strategies.

Botrytis squamosa, Botrytis aclada, and Sclerotium cepivorum are three fungal species of the family Sclerotiniaceae that are pathogenic on onion. Despite their close relatedness, these fungi cause very distinct diseases, respectively called leaf blight, neck rot, and white rot, which pose serious threats to onion cultivation. The infection biology of neck rot and white rot in particular is poorly understood. In this study, we used GFP-expressing transformants of all three fungi to visualize the early phases of infection. B. squamosa entered onion leaves by growing either through stomata or into anticlinal walls of onion epidermal cells. B. aclada, known to cause post-harvest rot and spoilage of onion bulbs, did not penetrate the leaf surface but instead formed superficial colonies which produced new conidia. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced appressorium-like structures and infection cushions, but upon prolonged contact with the non-host the infection structures died. With this study, we have gained understanding in the infection biology and strategy of each of these onion pathogens. Moreover, by comparing the infection mechanisms we were able to increase insight into how these closely related fungi can cause such different diseases.

​Bacterial Streak and Bulb Rot of Onion​

​Bacterial Streak and Bulb Rot of Onion​