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 (MLSA) 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. Pantoea 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 the second in aggressiveness on onion leaves, while 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.

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.

First Report of Onion (Allium cepa L.) 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 Pathogens Causing Bulb Rot Disease on Garlic (Allium sativum L.) in Central Java, Indonesia

Garlic bulb rot disease was found from garlics (Allium sativum L.) cultivated from 2017 to 2019 by farmers in Central Java Province, Indonesia. The initial symptoms of the disease were stunted, leaf yellowing, and necrotizing to rotten bulbs. This research was conducted to determine the major causal agent of garlic bulb rot disease in Central Java. A survey was carried out in five regencies across Central Java that were major garlic-producing areas. Nematodes were isolated using water immersion methode and pathogenic fungi were isolated on Potato Dextrose Agar (PDA). Nematode identification was carried out based on the Ditylenchus dipsaci morphological and morphometric character. Seven isolates of Fusarium species were obtained from infected garlic. Identification of four chosen isolates were performed by sequencing the TEF-1α gene. The TEF sequence of isolate TB3, KK1, and KK4 showed 99% similarity with several F. oxysporum, BT3 sequences showed 98% identity with several F. solani, and all were deposited in the NCBI GenBank. Three locations were positively infected by the interaction between D. dipsaci and Fusarium sp. Based on the results of the morphological identification, parasitic nematode was identified as D. dipsaci, while based on the morphological and molecular identification isolates Fusarium were identified as F. oxysporum and F. solani, respectively, as first report causal agents of garlic bulbs rot in Central Java.

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.

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.