Bakanae Research Articles

Whole Genome Sequencing of Fusarium fujikuroi Provides Insight into the Role of Secretory Proteins and Cell Wall Degrading Enzymes in Causing Bakanae Disease of Rice.

Fusarium fujikuroi causing bakanae disease has emerged as one of the major pathogen of rice across the world. The study aims to comparative genomic analysis of Fusarium fujikuroi isolates and identification of the secretary proteins of the fungus involved in rice pathogenesis. In the present study, F. fujikuroi isolate “F250” was sequenced with an assembly size of 42.47 Mb providing coverage of 96.89% on reference IMI58289 genome. A total of 13,603 protein-coding genes were predicted from genome assembly. The average gene density in the F. fujikuroi genome was 315.10 genes per Mb with an average gene length of 1.67 kb. Additionally, 134,374 single nucleotide polymorphisms (SNPs) are identified against IMI58289 isolate, with an average SNP density of 3.11 per kb of genome. Repetitive elements represent approximately 270,550 bp, which is 0.63% of the total genome. In total, 3,109 simple sequence repeats (SSRs), including 302 compound SSRs are identified in the 8,656 scaffolds. Comparative analysis of the isolates of F. fujikuroi revealed that they shared a total of 12,240 common clusters with F250 showing higher similarity with IMI58289. A total of 1,194 secretory proteins were identified in its genome among which there were 356 genes encoding carbohydrate active enzymes (CAZymes) capable for degradation of complex polysaccharides. Out of them glycoside hydrolase (GH) families were most prevalent (41%) followed by carbohydrate esterase (CE). Out of them CE8 (4 genes), PL1 (10 genes), PL3 (5 genes), and GH28 (8 genes) were prominent plant cell wall degrading enzymes families in F250 secretome. Besides this, 585 genes essential for the pathogen–host interactions were also identified. Selected genes were validated through quantitative real-time PCR analyses in resistant and susceptible genotypes of rice at different days of inoculation. The data offers a better understanding of F. fujikuroi genome and will help us enhance our knowledge on Fusarium fujikuroi–rice interactions.

Resistance mechanism of Fusarium fujikuroi to phenamacril in the field.

Rice bakanae disease, mainly caused by Fusarium fujikuroi, is an important disease of rice. Phenamacril has been used to control the disease for a few years in China. In 2016, nine phenamacril-resistant strains were found in the field in Zhejiang Province. The aim of the study was to clarify the mechanism of resistance of F. fujikuroi to phenamacril and the fitness of resistant strains. The nine F. fujikuroi strains examined were highly resistant to phenamacril. Eight of them had the point mutation TCA (Ser) → CCA (Pro) at codon 219 in the Myosin-5 protein, while the other had the point mutation TCA (Ser) → TTA (Leu) at codon 219. Myosin-5 replacement between resistant and sensitive strains confirmed that the point mutation in Myosin-5 caused the resistance of F. fujikuroi to phenamacril. Docking of phenamacril into the modeled binding pocket of Myosin-5 showed that the affinity between phenamacril and Myosin-5 decreased and a hydrogen bond could not be formed between phenamacril and the amino acid at codon 219 after it changed to Pro or Leu. There was no cross-resistance between phenamacril and other fungicides. The eight resistant strains containing the point mutation S219P had almost the same fitness as the sensitive strains, while the one resistant strain containing the point mutation S219 L showed decreased mycelial growth, sporulation and pathogenicity. In the field, the point mutation S219P or S219 L in Myosin-5 conferred high resistance to phenamacril in F. fujikuroi. The point mutation S219P did not affect the fitness of F. fujikuroi, while the point mutation S219 L decreased its fitness. © 2017 Society of Chemical Industry.

Identification of bakanae disease resistance loci in japonica rice through genome wide association study

BackgroundBakanae disease, caused by seed-borne Fusarium species, mainly F. fujikuroi, is a rice disease whose importance is considerably increasing in several rice growing countries, leading to incremental production losses.ResultsA germplasm collection of japonica rice was screened for F. fujikuroi resistance, allowing the identification of accessions with high-to-moderate levels of resistance to bakanae. A GWAS approach uncovered two genomic regions highly associated with the observed phenotypic variation for response to bakanae infection on the short arm of chromosome 1 (named as qBK1_628091) and on the long arm of chromosome 4 (named as qBK4_31750955). High levels of phenotypic resistance to bakanae were associated to the cumulated presence of the resistant alleles at the two resistance loci, suggesting that they can provide useful levels of disease protection in resistance breeding. A fine comparison with the genomic positions of qBK1_628091 and qBK4_31750955 with respect to the QTLs for bakanae resistance reported in the literature suggests that the resistant loci here described represent new genomic regions associated to F. fujikuroi resistance. A search for candidate genes with a putative role in bakanae resistance was conducted considering all the annotated genes and F. fujikuroi-related DEGs included in the two genomic regions highlighting several gene functions that could be involved in resistance, thus paving the way to the functional characterization of the resistance loci.ConclusionsNew effective sources for bakanae resistance were identified on rice chromosomes 1 and 4 and tools for resistance breeding are provided.

Effect of Rice Seed Disinfection of Loess-sulfur on the Suppression of Bakanae disease caused by Fusarium fujikuroi

Effect of Rice Seed Disinfection of Loess-sulfur on the Suppression of Bakanae disease caused by Fusarium fujikuroi

Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds.

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

Effect of Silicate-Coated Rice Seed on Healthy Seedling Development and Bakanae Disease Reduction when Raising Rice in Seed Boxes

Effect of Silicate-Coated Rice Seed on Healthy Seedling Development and Bakanae Disease Reduction when Raising Rice in Seed Boxes

First report of Fusarium fujikuroi in the Lao PDR

The isolation of Fusarium fujikuroi from rice plants with elongated stems typical of bakanae disease in the Lao PDR is reported for the first time. The identification was based on both molecular and morphological markers. Koch’s postulates were fulfilled. Bakanae disease has long been recognized in the Lao PDR based on symptomatology, but the etiology of the disease has not been clearly defined. This study was part of a continuing program to assist with the development of checklists of pathogens and plant diseases for Lao PDR for biosecurity purposes and integrated disease management.

The puzzle of bakanae disease through interactions between Fusarium fujikuroi and rice.

Bakanae disease, one of the most noteworthy seedborne rice diseases, is caused by Fusarium fujikuroi, a member of the Gibberella fujikuroi species complex. The decreasing availability of chemical seed-dressing products over the last few years has raised the concerns of rice seed companies regarding bakanae disease. Therefore, new research trends require a deeper investigation into the main aspects of bakanae disease through interactions between rice and F. fujikuroi, in order to find new resistant or tolerant cultivars and alternative bakanae disease control strategies, as well as to develop more sensitive molecular diagnostic techniques. Here, some new aspects of F. fujikuroi epidemiology and pathogenicity, as well as its interactions with rice, are reported, and recent approaches applied to control bakanae disease are summarized.

Influence of Cold Condition during the Greening Period to Occurrence of Bakanae Disease

Influence of Cold Condition during the Greening Period to Occurrence of Bakanae Disease

Evaluation of resistance sources against foot rot and bakanae disease of basmati rice

Evaluation of resistance sources against foot rot and bakanae disease of basmati rice

SINGLE AND COMBINED EFFECTS OF THREE FUSARIUM SPECIES ASSOCIATED WITH RICE SEEDS ON THE SEVERITY OF BAKANAE DISEASE OF RICE

Fusarium spp. associated with susceptible rice variety Pusa Basmati 1121 were isolated. Based on translation elongation factor 1α (TEF-1α) Fusarium spp. were identified as F. fujikuroi, F. proliferatum, F. verticillioides (F. moniliforme), F. sacchari, F. pseudonygamai, F. thapsinum and F. andiyazi. Pathogenicity test conducted on susceptible variety Pusa Basmati 1121 confirmed that only F. fujikuroi, F. proliferatum and F. verticillioides produced bakanae disease as elongation, rotting or both types of symptoms. Gibberellic acid was exclusively extracted from the isolates of F. fujikuroi and it ranged from 450 μg/g to 4361 μg/g of mycelium. Gibberellic acid content was positively correlated (r=0.79) with elongation symptom of the disease. Fusaric acid was present in all the species of Fusarium, ranging from 60 μg/g to 2920 μg/g of mycelium. However, significant correlation between fusaric acid content and disease severity could not be established. Co-inoculations of pathogens showed significant reduction in percent seed germination and increased disease severity compared to pathogen inoculated individually. Therefore, present investigation will be helpful to understand the effect of different Fusarium spp. in bakanae disease etiology.

Mapping quantitative trait loci responsible for resistance to Bakanae disease in rice.

BackgroundBakanae or foot rot disease caused by Fusarium fujikuroi [teleomorph: Gibberella fujikuroi (Sawada) Ito] is emerging as a serious disease in rice. The disease causes both quantitative and qualitative losses to the grains under the field conditions. Breeding for resistance to Bakanae disease is a promising strategy to manage this emerging disease. In this study, we used a population of 168 F14 recombinant inbred lines (RILs) derived from two indica rice parents Pusa 1342, a highly resistant variety and Pusa Basmati 1121, a highly susceptible variety to map quantitative trait loci (QTLs) governing resistance against Bakanae disease.ResultsThe disease reaction of 168 F14 RILs were measured on the seedlings inoculated using Fusarium fujikuroi culture using high-throughput screening protocol under glasshouse conditions. Utilizing inclusive composite interval mapping, three QTLs governing resistance to Bakanae were identified, namely qBK1.1, qBK1.2 and qBK1.3 which accounted 4.76, 24.74 and 6.49 % of phenotypic variation, respectively. The major effect QTL designated qBK1.2 was mapped in 0.26 Mb region between RM5336 and RM10153. A total of 55 annotated genes were identified within the identified QTL region qBK1.2.ConclusionsThe novel QTLs identified in this study are useful resource for efficiently breeding rice cultivars resistant to Bakanae disease. This is the first report on identification of QTLs governing resistance against Bakanae in rice using inclusive composite interval mapping strategy in a RIL population.

Transcriptomic dissection of the rice–Fusarium fujikuroi interaction by RNA-Seq

Rice bakanae disease, caused by the pathogen Fusarium fujikuroi, is becoming severely detrimental to rice production worldwide. To understand the interaction between rice and F. fujikuroi, a moderate resistant genotype, 93-11 (Oryza sativa indica), and a susceptible genotype, Nipponbare (Oryza sativa japonica), were used for transcriptome analysis. Several cDNA libraries were constructed using mRNA isolated from the leaves of non-stressed 93-11 and NIPPONBARE plants and from the leaves of both genotypes inoculated with F. fujikuroi. In total, 1152 and 1052 transcripts were differentially expressed between the controls and the treatments for 93-11 and NIPPONBARE, respectively. Comparative transcriptome analysis revealed different expression patterns for the two genotypes. Although some common defense-related enriched GO terms were shared in both genotypes, specific defense-related terms were enriched exclusively in 93-11. A detailed comparison of defense-related differentially expressed genes revealed that certain WRKYs, WAK and MAP3Ks were responsible for the bakanae disease resistance in 93-11. The OsWAK112d gene was up-regulated in the resistant genotype. The POEI gene response to abiotic stress was modulated in Nipponbare. Further analysis suggested that the defense-related genes WRKYs and MARKs on chromosome 1 that are modulated in 93-11 upon infection might play a crucial role in the rice–F. fujikuroi interaction. Further characterization of these resistance genes may provide candidate genes for the development of molecular markers for rice bakanae resistance breeding programs. Transcriptomic dissection of the rice–F. fujikuroi interaction provides valuable information for future studies on the molecular mechanisms of rice bakanae resistance.

Management of bakanae and bacterial seedling blight diseases in nurseries by irradiating rice seeds with atmospheric plasma

The control of seedborne rice seedling diseases in the seed beds is important to avoid epidemics in rice nurseries and paddies, which may result in severe yield loss. Recently, irradiation with plasma containing electrons, creating positive or negative ions and neutral species, has been shown to have an antimicrobial effect, probably via generation of reactive oxygen species. This study examines whether two seedborne rice seedling diseases, bakanae disease caused by the fungal pathogen Fusarium fujikuroi, and bacterial seedling blight caused by Burkholderia plantarii, are suppressed by irradiation of infected rice seeds with atmospheric plasma. Seed germination and seedling growth were not inhibited in plasma‐treated healthy seeds. When F. fujikuroi‐infected rice seeds were irradiated with plasma after being immersed in sterile distilled water, bakanae disease severity index and the percentage of plants with symptoms were reduced to 18.1% and 7.8% of non‐irradiated control, respectively, depending on the duration of plasma irradiation. The bacterial seedling blight disease index was also reduced by plasma irradiation in vacuum‐inoculated seeds to 38.6% of the non‐irradiated control, and in infected seeds harvested from spray‐inoculated heads of rice plants to 40.1% of the control. Therefore, plasma irradiation seems to be effective in controlling two independent seedborne rice seedling diseases.

Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi.

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

Biological Control of Rice Bakanae by an Endophytic Bacillus oryzicola YC7007.

In our previous study, we reported that a novel endophytic bacterium Bacillus oryzicola YC7007 has suppressed bacterial diseases of rice via induced systemic resistance and antibiotic production. This endophytic strain, B. oryzicola YC7007 was used as a biological control agent against bakanae disease of rice caused by Fusarium fujikuroi, and its mechanism of interaction with the pathogen and the rice was further elucidated. Root drenching with B. oryzicola YC7007 suspension reduced the disease severity of bakanae significantly when compared with the untreated controls. The treatments of B. oryzicola YC7007 suspension (2.0 × 107 cfu/ml) to the rice rhizosphere reduced bakanae severity by 46–78% in pots and nursery box tests containing autoclaved and non-autoclaved soils. Moreover, in the detached rice leaves bioassay, the development of necrotic lesion and mycelial expansion of F. fujikuroi were inhibited significantly by spraying the culture filtrate of B. oryzicola YC7007. Drenching of ethyl acetate extracts of the culture filtrate to the rhizosphere of rice seedlings also reduced the bakanae disease severity in the plant culture dish tests. With the root drenching of B. oryzicola YC7007 suspension, the accumulation of hydrogen peroxide was observed at an early stage of rice seedlings, and a hormonal defense was elicited with and without pathogen inoculation. Our results showed that the strain B. oryzicola YC7007 had a good biocontrol activity against the bakanae disease of rice by direct inhibition, and was also capable of inducing systemic resistance against the pathogen via primed induction of the jasmonic acid pathway.

Incidence of Bakanae disease of basmati rice in south-western part of Punjab and its management

Basmati rice is an important cash crop of India, being recognized at international level for its quality and aroma. Bakanae/foot rot disease is becoming a limiting factor in its cultivation in southwestern parts of Punjab. A survey was conducted during Kharif 2014 in different blocks of Sri Muktsar Sahib District of Punjab to acquaint with the factor affecting disease incidence. Disease was recorded in all the basmati growing fields and up to 10 per cent incidence was recorded on different aromatic rice cultivars. Pusa Basmati 1401 was observed more susceptible followed by Pusa Basmati 1121 and Pusa Basmati 1509.Basmati crop raised at recommended period showed lower disease incidence than early transplanted crop. Higher disease incidence was observed in fields where seed was purchased from private seed companies than seed from State Agricultural University. The recommended technology to manage this disease i.e. seed soaking in pesticides solution containing carbendazim 50 WP @ 0.2 per cent + Streptocycline 0.01 per cent (one g) for 12 hrs followed by seedling root dip in carbendazim 50 WP (0.2%) for 6 hrs was demonstrated at selected farmers’ field during Kharif 2015. The results showed that seed treatment with carbendazim @ 2.0 g/kg alone (farmers’ practice) was not effective. The disease incidence in demonstrated plots was less as compared to farmers’ practice. The grain yield and net returns were also higher in demonstrated plots. How to view point the article : Sandhu, Gurmail Singh and Dhaliwal, N.S. (2016). Incidence of Bakanae disease of basmati rice in south-western part of Punjab and its management. Internat. J. Plant Protec., 9(1) : 353-357.

Screening of Rice Germplasm for Bakanae Disease Resistance in Rice

Screening of Rice Germplasm for Bakanae Disease Resistance in Rice

Bakanae Disease Reduction Effect by Use of Silicate Coated Seed in Wet Direct-Seeded Rice

벼 직파재배에서 볍씨에 피복재인 지오라이트와 결합재인 수용성규산(25%)으로 혼합 코팅된 "규산코팅볍씨(SCS)"의 활용에 의한 키다리병 억제효과를 구명하고자 서해안 간척지유래토양에서 담수표면직파된 포장시험과 아울러 시험포장에서 발병된 키다리병 이병주를 접종원으로 하여 변온조건(주야 <TEX>$18{\sim}26^{\circ}C$</TEX>)과 항온조건(<TEX>$30^{\circ}C$</TEX>)에서 실내시험으로 수행한 결과는 다음과 같다. 1. 담수직파에서 규산코팅볍씨 처리구는 무코팅볍씨 처리구에서 보다 입모분포상태가 균일하였고 분얼중기경 건물생산량이 13% 증가되었다. 2. 무코팅볍씨 처리구에서는 분얼중기 경부터 키다리병이 발생되어 종자감염을 일으키는 출수기 경에 9.9%의 심한 이병경율을 보인 반면에 규산코팅볍씨 처리구에서는 분얼중기까지 거의 발생이 없다가 출수기 무렵에 0.01%의 극미한 발생으로 99.9%의 방제가를 보였다. 이병주의 줄기마디와 이삭영화 감염부위에서는 전형적인 키다리병균의 소형분생자와 대형분생자 및 균사체 등이 확인되었다. 3. 무코팅볍씨와 규산코팅볍씨를 <TEX>$30^{\circ}C$</TEX> 항온기에 각각 치상하고 시험포장에서 발병된 이병주의 이삭을 2주일간 접종 처리한 결과 무코팅볍씨 처리구의 접종이삭 종자에서는 종자표면에 균총이 형성되었고 종자내에는 소형분생자, 대형분생자 및 균사 발육이 왕성하게 증식된 반면에 규산코팅볍씨 처리구에서는 소형분생자와 균사는 생존하지 못하였고 대형분생자와 균사체 발육은 크게 위축되었다. 이상의 결과로 규산코팅볍씨의 활용으로 직파재배 시 종자소독 없이도 친환경적 키다리병 방제가 가능하며, 향후 종자소독제로의 개발도 가능할 것으로 판단되었다. To investigate the effect of soluble silicate zeolite dressing of the rice against bakanae disease, field trial in reclaimed land and in vitro were carried out. The coated rice seeds (SCS) which were dressed with the mixture of 25% silicic acids (binder), and the zeolite (coating powder). In wet direct seeding, uniform scattering of rice seeds on the soil surface and the better seedling establishment were shown in SCS treatment plots. The incidence of bakanae disease began from the mid tillering stage toward the heading stage. Around heading stage, the ratio of infected tillers reached its highest point by 9.9% in non-SCS treatment plots. While, in SCS treatment plots, the ratio of infected tillers was no more than 0.01%. The vitality of the pathogenic fungi of bakanae disease in the SCS and non-SCS samples were assessed. Samples were incubated for one week keeping proper humidity at <TEX>$30^{\circ}C$</TEX> after inoculated with panicles of infected rice plants from experimental field plots. In non-SCS treatment, pinkish colonies were formed on the grain surface of panicle of infected plants, and mycelium, macro-conidia and micro-conidia were developed actively inside part of infected grain inoculated. While in SCS treatment, micro-conidia and mycelium were not survived and the growth of macro-conidia, mycelia were greatly inhibited and withered. Based on the results, it is concluded that the environmental friendly control of bakanae disease by use of SCS is possible and soluble silicate can be applied as agents for replacement of seed disinfection.

Detecting Bakanae disease in rice seedlings by machine vision

Bakanae disease, or “foolish seedling”, is a seed-borne disease of rice (Oryza sativa L.). Infected plants can yield empty panicles or perish, resulting in a loss of grain yield. The disease occurs most frequently when contaminated seeds are used. Once the seeds are contaminated, the pathogen Fusarium fujikuroi spreads in the field. Therefore, infected plants must be screened at early developmental stages. This work proposes an approach to nondestructively distinguish infected and healthy seedlings at the age of 3weeks using machine vision. Seeds of the rice cultivars Tainan 11 and Toyonishiki were inoculated with a conidial suspension of F. fujikuroi. The seedling were cultivated in an incubator for 3weeks. The images of infected and control seedlings were acquired using flatbed scanners to quantify their morphological and color traits. Support vector machine (SVM) classifiers were developed for distinguishing the infected and healthy seedlings. A genetic algorithm was used for selecting essential traits and optimal model parameters for the SVM classifiers. The proposed approach distinguished infected and healthy seedlings with an accuracy of 87.9% and a positive predictive value of 91.8%.