First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch’s postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

Genomic, Antimicrobial, and Aphicidal Traits of Bacillus velezensis ATR2, and Its Biocontrol Potential against Ginger Rhizome Rot Disease Caused by Bacillus pumilus

Ginger rhizome rot disease, caused by the pathogen Bacilluspumilus GR8, could result in severe rot of ginger rhizomes and heavily threaten ginger production. In this study, we identified and characterized a new Bacillus velezensis strain, designated ATR2. Genome analysis revealed B. velezensis ATR2 harbored a series of genes closely related to promoting plant growth and triggering plant immunity. Meanwhile, ten gene clusters involved in the biosynthesis of various secondary metabolites (surfactin, bacillomycin, fengycin, bacillibactin, bacilysin, difficidin, macrolactin, bacillaene, plantazolicin, and amylocyclicin) and two clusters encoding a putative lipopeptide and a putative phosphonate which might be explored as novel bioactive compounds were also present in the ATR2 genome. Moreover, B. velezensis ATR2 showed excellent antagonistic activities against multiple plant pathogenic bacteria, plant pathogenic fungi, human pathogenic bacteria, and human pathogenic fungus. B. velezensis ATR2 was also efficacious in control of aphids. The antagonistic compound from B. velezensis ATR2 against B.pumilus GR8 was purified and identified as bacillomycin D. In addition, B. velezensis ATR2 exhibited excellent biocontrol efficacy against ginger rhizome rot disease on ginger slices. These findings showed the potential of further applications of B. velezensis ATR2 as a biocontrol agent in agricultural diseases and pests management.

Ginger Rhizome Rot caused by the Enterobacter cloacae in Tangshan, China

Ginger (Zingiber officinale ROSC.) is an important economic crop in China, especially the rhizome tissue has a high medicinal value. In July 2019, the symptom of ginger rhizome rot appeared in Tangshan city of Hebei Province, with incidence rates of 15%. The diseased part of ginger rhizome became soft and presented light brown maceration. Serious internal all erosion, only have epidermis, gray-white juice overflowed the epidermis, and with foul smell. The surface of ginger rhizome was disinfected with 1% NaOCl, and colonies were isolated and purified on nutrient agar (NA) solid medium by streak plate method. Eight isolates were obtained from 15 diseased tissue samples. Further morphological, physiological and biochemical identification of the pure cultured bacteria were carried out. Two isolates of bacteria were picked for further analysis. The bacteria were gram-negative bacilli, which were milky white and round protuberances on NA solid medium. Physiological and biochemical test results showed that isolates were facultatively anaerobic, gelatin liquefaction; negative for indole, methyl red, hydrogen sulfide production and the Voges-Prauskauer test (V-P); positive for D-glucose, sucrose, sorbitol, inositol, mannitol and citrate utilization. A typical hypersensitive reaction was induced on 12-week-old tobacco (Nicotiana benthamiana) leaves, which were inoculated by injecting suspensions of the isolated strain (108 CFU/mL) at 25℃ after 24-h. These characteristics were consistent with Enterobacter spp. To further assess the identity of the species, the genomic DNA was extracted from one bacterium（SDXJ1）. The partial 16S rRNA gene and specific rpoB and gyrB genes were amplified and sequenced with primers 27F/1492R, CM7/CM31b and UP1f/UP2r (Brady et al. 2013, Mollet et al. 1997, Lane 1991). The obtained 16S and rpoB sequences (GenBank accession MK937637, MZ911902 and MZ911901) of the isolate showed 99.33%, 99.21% and 99.57% identity to the corresponding sequences of Enterobacter cloacae in GenBank (CP055458, AP022228 and AP022519). Maximum likelihood analysis was performed, and the phylogenetic tree clustered with E. cloacae (MEGAX, Bootstrap n=1000). The pathogenicity of the isolates was tested on ginger plants and rhizomes tissue. The bacterial suspensions (108 CFU/mL) of two isolates were injected into the basal stem and rhizomes center of 6 healthy ginger seedlings respectively, and control groups were treated with sterile water. The inoculated plants were kept in a moist chamber (28°C, 16-h light and 8-h dark period) and ginger rhizomes were placed in the incubator (30°C, 16-h light and 8-h dark period). Seven days after inoculation, the ginger tubers showed symptoms of decay, and 20 to 25 days later, the ginger plant leaves browned and died. The pathogenicity test was repeated 4 times and all controls were healthy. The pathogens of symptomatic plants and ginger rhizomes were studied. They were identified as E. cloacae by physiological, biochemical and molecular biological methods, fulfilling Koch's hypothesis. This is the first report of the ginger rhizome rot caused by the Enterobacter cloacae in Tangshan, China. The research results are of great significance for the prevention and control of the disease. Our laboratory has reported that Citrobacter freundii can cause Ginger Rot. (Zhao et al. 2021) Whether the two strains infect alone or compound in the field still needs to be further explored.

ANALISIS PENDAPATAN USAHATANI JAHE (ZINGIBER officinale) Di Kecamatan Liang Anggang Kota Banjarbaru Provinsi Kalimantan Selatan

The research aimed to determine the explicit cost, revenue, income, and feasibility obtained from ginger farming. This research was conducted for 3 months from June 2020 to September 2020 in Liang Anggang District Banjarbaru City South Kalimantan Province. This research uses a case study method of farmers who do ginger farming, which only has one person in this research location. From this research, it was found that the total explicit cost is Rp. 66.475.000, total revenue is Rp. 158.525.000 and the total income is Rp. 225.000.000. Also ginger is a plant that deserves to be cultivated because R/C > 1, which is 3.38, which means that for every Rp. 100, - issued, you will receive an income of Rp. 338,-. And efforts that can be made to increase income are by increasing the area of ginger plants and intensive care to prevent rhizome rot to increase ginger production

Integration of physical approach and chemical approach for the control of banana rhizome rot in first and ratoon crop of banana cv. KovvurBontha (ABB)

India is the major producer of the banana crop in the world. Banana is divided into two types. First one is desert type and the second one is the cooking type of banana. Cooking banana is the stapple food in many parts of the India especially in Kerala. Major problem in cooking variety is that these varieties are highly susceptible to rhizome rot disease. To control this integration of physical method (collection and destruction) and chemical method (application of fungicide and bactericide) was used at an interval of 7 days and 14 days. The results showed that combination of physical method and application of MetalaxylMz (4 mgL-1) + streptomycin (1.5 mg/10L) at 7 days interval had resulted in survival percentage (96.03%), average number of the fingers/hand (12.67), average number of the hands/bunch (9.27), average weight of the bunch (29.67 kg) and yield per hectare (61.58 t/ha) in first crop. on the other hand, ratoon crop had recorded maximum survival percentage (99.83%), maximum average number of the fingers/hand (13.00), maximum average number of the hands/bunch (8.53), maximum average weight of the bunch (28.41kg) and maximum yield per hectare (59.45t/ha). This experiment concluded that rhizome rot can be completely control by the integrated physical and chemical method of disease management.

LAMP assay for distinguishing Fusarium oxysporum and Fusarium commune in lotus (Nelumbo nucifera) rhizomes

The yields of edible rhizome from the cultivation of the perennial hydrophyte lotus (Nelumbo nucifera) can be severely reduced by rhizome rot disease caused by Fusarium species. There is a lack of rapid field-applicable methods for detection of these pathogens on lotus plants displaying symptoms of rhizome-rot. Fusarium commune (91%) and Fusarium oxysporum (9%) were identified at different frequencies from lotus samples showing symptoms of rhizome-rot. As these two species can cause different severity of disease and their morphology is very similar, molecular-diagnostic based methods to detect these two species were developed. Based on the comparison of the mitochondrial genome of the two species, three specific DNA loci targets were found. The designed primer sets for conventional PCR, qPCR and loop-mediated isothermal amplification (LAMP) precisely distinguished the above two species when isolated from lotus and other plants. The LAMP detection limits were 10 pg/μl and 1 pg/μl of total DNA for F. commune and F. oxysporum, respectively. We also carried out field-mimicked experiments on lotus seedlings and rhizomes (including inoculated samples and field diseased samples), and the results indicated that the LAMP primer sets and the supporting portable methods are suitable for the rapid diagnosis of the lotus disease in the field. The LAMP-based detection method will aid in the rapid identification of whether F. oxysporum or F. commune are infecting lotus plants with symptoms of rhizome-rot, and can facilitate efficient pesticide use and prevent the disease spread through vegetative propagation of Fusarium-infected lotus rhizomes.

Efficacy of Different Fungicides Against Rhizome Rot Disease of Ginger Under Farmer Field Condition in Salyan, Nepal

A study was conducted from March to August 2020 to evaluate the effectiveness of different fungicides for the management of rhizome rot disease in ginger. The field experiment was conducted under Randomized Complete Block Design with four replication and five treatments; Carbendazim 50% WP @ 1gm/litre + Mancozeb 75% WP @ 2.5gm/litre, Metalaxyl 8% WP + Mancozeb 64% WP @ 2gm/litre, Carbendazim 50% WP @ 1gm/litre, Trichoderma viride @ 5gm/litre and control. The height of pseudo stems, leaf number, germination percentage, tiller number, disease incidence, disease severity and percentage of disease control was recorded during field experiment. There was no significant effect of pre sowing rhizome treatment in height of pseudostems, leaf number and germination percentage. The highest tiller number (3.40) was found with Carbendazim 50% WP @1gm/litre + Mancozeb 75% @ 2.5gm/ litre treatment and lowest tillers number (2.95) was found with control. The highest disease incidence (34.37) and severity (30.03) was found in control plot whereas the lowest disease incidence (2.58) and disease severity (2.46) was found with Carbendazim 50% @ 1gm/litre treatment + Mancozeb 75% @ 2.5gm/litre. Similarly, the highest percentage disease control (85.61) was found with Carbendazim 50% @ 1gm/litre + Mancozeb 75% @ 2.5gm/litre treated rhizome and the lowest (54.14) was found with Trichoderma viride @ 5gm/litre treatment. Therefore, seed treatment with Carbendazim 50%WP @ 1gm/litre + Mancozeb 75% WP @ 2.5 gm/litre is found effective among other fungicides in Bagchaur-3, Salyan and is recommended for enhancing tiller and reducing rhizome rot disease incidence and severity in ginger.

Microbial inoculants and Trichoderma viride consortia for growth promotion and disease management in ginger

A study was conducted to evaluate the effect of microbial inoculants consortia on ginger under field conditions. Three biofertilizers comprising of Azospirillum lipoferum (nitrogen fixer KAU-AZO), Bacillus megaterium (phosphate solubilizer KAU-PSB), Bacillus sporothermodurans (potash solubilizer KAU-KSB) and two biocontrol agents namely, Pseudomonas fluorescen (KAU-PF) and Trichoderma viride (KAU-TV) were used in the study. Compatibility studies revealed that all the isolates were compatible with each other. However, P. fluorescens and T. viride were incompatible with each other under in vitro. Under field evaluation, consortia of microbial inoculants performed better than the single inoculants in terms of yield and disease management. The organic Adhoc package recorded the minimum incidence of rhizome rot (2.09 %) among all the treatments. However, KAU-AZO + KAU-PSB + KAU-KSB + KAU-TV recorded the minimum rhizome rot (5.23%) incidence among the consortia. In the case of Rhizoctonia leaf blight disease, consortia of KAU-AZO + KAU-PSB + KAU-KSB + KAU-PF showed the minimum incidence (5.21%). In general, consortium of A. lipoferum, B. megaterium (PSB), B. sporothermodurans (KSB) and T. viride was the most efficient for plant growth promotion and disease management in ginger.

First Report of Fusarium Yellows and Rhizome Rot Caused by Fusarium oxysporum f. sp. zingiberi on Ginger in the Continental United States

Ginger (Zingiber officinale Roscoe) is one of the most widely consumed medicinal herb in the world, and the U.S. imports of ginger have risen in recent years because of its health benefits. Seed rhizome and soilborne diseases are serious concerns of ginger worldwide (Stirling 2004; Moreira et al. 2013), including the recent observations of Fusarium yellows and rhizome rot in the Commonwealth of Virginia. In October 2018 and 2019, ginger plants with yellowing of leaf margins and stunted growth were uprooted from a 9.1 m × 14.6 m high tunnel (HT) and from an outdoor raised bed at Virginia State University’s Randolph farm. Disease incidence in the HT and the raised bed was estimated between 5-70%. Small pieces (2-5 mm) of symptomatic rhizomes were disinfected with 0.6% sodium hypochlorite solution and placed on potato dextrose agar (PDA) Petri plates to recover fungal isolates. Hyphal tips from these isolates were transferred to fresh PDA to obtain pure cultures. The fungal colonies were pinkish-white initially, and turned purplish-pink after 5-7 days of incubation at 25 °C. The microconidia were aseptate, oval or elliptical, hyaline, and measured 5 to 12 × 4 to 6 µm in size. Macroconidia were with 3 to 5 septations, curved like a sickle towards the ventral side, hyaline, smooth and thin-walled, and 15 to 40 × 3 to 6 µm in size. Fungal genomic DNA of one isolate (Gf-VA-3) was extracted from a 7-days old culture using PrepMan®Ultra (Thermo Fischer Scientific, Cheshire UK). Four conserved regions of the isolated pathogen, internal transcribed spacer (ITS), translation elongation factor (EF), β-tubulin (Bt), and calmodulin (cal) gene regions were amplified using ITS1 and ITS4 (White et al. 1990), ef1α and ef2α (O’Donnell et al. 1998), Bt2a and Bt2b (Glass and Donaldson 1995), and calA1 and calQ1 (Carbone and Kohn 1999), respectively. PCR products were sequenced, and amplicons deposited in GenBank with accession numbers MT337417 for ITS, MT436712 for Bt, MT802441 for cal and MW816632 for EF. A 99-100% identity with Fusarium oxysporum was matched with accession nos. MW776326 for ITS, MN646766 for the β-tubulin, MT010904 for the calmodulin and MN258350 for the translation elongation factor genes. For pathogenicity test, six 6-week-old healthy ginger plants grown on sterilized potting mix in the greenhouse were inoculated by injecting 3-ml of a 1 × 108 micro- and macro-conidia suspension per ml at the crown area transcending to the rhizome. Another set of six plants were injected with distilled and autoclaved water in the same way. After four weeks, leaves withered, plants exhibited yellowing and wilt followed by stunted growth and eventually complete collapse of the six inoculated plants, however control plants showed none of the symptoms. The same pathogen was re-isolated from the inoculated plants. The pathogenicity test was repeated, and the same results were observed. Fusarium yellows and rhizome rot has been reported from Hawaii in the U.S. (Trujillo 1963), Brazil (Moreira et al. 2013), Australia (Stirling 2004), China (Li et al. 2014), and India (Shanmugam et al. 2013). To our knowledge, this is the first report of Fusarium yellows and rhizome rot on ginger in the Continental U.S. The disease is seed rhizome and soilborne leading to poor establishment and hence economic loss in ginger production