Pokkah Boeng Research Articles

Fusarium sacchari Effector FsMEP1 Contributes to Virulence by Disturbing Localization of Thiamine Thiazole Synthase ScTHI2 from Sugarcane

Fusarium sacchari is a significant pathogenic fungus that causes sugarcane Pokkah Boeng. Proteins secreted by pathogenic fungi can be delivered into hosts to suppress plant immunity and establish infection. However, there is still much to be discovered regarding F. sacchari’s secreted effectors in overcoming plant immunity. In this paper, we characterize a novel effector called FsMEP1, which is essential for the virulence of F. sacchari. FsMEP1 contains a conserved zinc-binding motif sequence, HEXXH, and is highly expressed during host infection. Using the Agrobacterium tumefaciens-mediated transient expression system, it was confirmed that FsMEP1 could suppress Bcl-2-associated X protein (BAX)-triggered cell death, callose deposition, and ROS explosion in Nicotiana benthamiana. Furthermore, the deletion of FsMEP1 demonstrated its requirement for contributing to the pathogenicity of F. sacchari in sugarcane. Further analysis revealed that FsMEP1 could interact with the sugarcane thiamine thiazole synthase ScTHI2 and disrupt its normal localization, thereby inhibiting the synthesis of thiamine and the defense responses mediated by ScTHI2. Based on these findings, we propose that ScTHI2 represents a potential molecular target for improving sugarcane resistance to Pokkah Boeng disease.

Identification of Reactive Oxygen Species Genes Mediating Resistance to Fusarium verticillioides in the Peroxisomes of Sugarcane

Pokkah boeng disease (PBD), which is caused by Fusarium verticillioides, is a major sugarcane disease in Southeast Asian countries. Breeding varieties to become resistant to F. verticillioides is the most effective approach for minimizing the damage caused by PBD, and identifying genes mediating resistance to PBD via molecular techniques is essential. The production of reactive oxygen species (ROSs) is one of a cell’s first responses to pathogenic infections. Plant peroxisomes play roles in several metabolic processes involving ROSs. In this study, seedlings of YT94/128 and GT37 inoculated with F. verticillioides were used to identify PBD resistance genes. The cells showed a high degree of morphological variation, and the cell walls became increasingly degraded as the duration of the infection increased. There was significant variation in H2O2 accumulation over time. Catalase, superoxide dismutase, and peroxidase activities increased in both seedlings. Analysis of differentially expressed genes (DEGs) revealed that peroxidase-metabolism-related genes are mainly involved in matrix protein import and receptor recycling, adenine nucleotide transport, peroxisome division, ROS metabolism, and processes related to peroxisomal membrane proteins. The expression levels of SoCATA1 and SoSOD2A2 gradually decreased after sugarcane infection. F. verticillioides inhibited the expressions of C5YVR0 and C5Z4S4. Sugarcane infection by F. verticillioides disrupts the balance of intracellular ROSs and increases the cell membrane’s lipid peroxidation rate. Defense-related enzymes play a key regulatory role in maintaining a low, healthy level of ROSs. The results of this study enhance our understanding of the mechanism through which peroxisomes mediate the resistance of sugarcane to PBD and provide candidate genes that could be used to breed varieties with improved traits via molecular breeding.

A dual-mode strategy for early detection of sugarcane pokkah boeng disease pathogen: A portable sensing device based on Cross-N DNA framework and MoS2@GDY

Sucrose, a common sugar primarily derived from sugarcane, is a crucial national strategic resource. However, its yield is significantly affected by various serious diseases, with pokkah boeng disease being one of the most damaging. Therefore, developing a sensitive method for the accurate detection of the pokkah boeng disease pathogen is crucial for ensuring the safety of sugar. This work presents a portable dual-modal detection device, assisted by a smartphone, which is based on MoS2@GDY, Mn3O4@Au nanomenzyme, cross-N DNA framework and Exo III exonuclease-assisted CHA signal amplification technology. The cross-N DNA framework provides many binding sites and is not restricted by AuNPs scattering positions, enhancing the signal output strength of the sensor. Additionally, the detection system incorporates a high-power-density capacitor to further amplify the electrochemical detection signal, increasing sensitivity by 9.1 times. Moreover, the use of electrochemical and colorimetric dual-mode detection effectively avoids mutual interference, reducing the likelihood of false positives from a single signal. Under optimized conditions, the proposed method has a linear range of 0.0001–10,000 pM, and with a detection limit of 6.1 aM (S/N=3). This high-sensitivity, high-reliability portable sensing method shows significant potential for the early detection and real-time on-site monitoring of the pokkah boeng disease pathogen.

Endophyte Chaetomium Globosum CGSR-13 strain enhanced plant growth promotion and antifungal activity against pokkah boeng caused by Fusarium verticillioides in India

Endophyte Chaetomium Globosum CGSR-13 strain enhanced plant growth promotion and antifungal activity against pokkah boeng caused by Fusarium verticillioides in India

Dual-electrode signal amplification self-powered biosensing platform based on nanozyme boosting target-induced DNA nanospace array for ultrasensitive detection of sugarcane Pokkah Boeng disease pathogenic bacteria

Sugarcane is a crop with significant economic importance worldwide. However, pokkah boeng disease poses a serious threat to its production and the sustainable development. There is a pressing necessity for precise and portable detection methods. We develop a dual-electrode signal amplification biosensing platform, for highly sensitive detection of sugarcane pokkah boeng disease pathogenic bacteria. This innovative platform integrates highly catalytic AuNPs/Mn3O4 nanozymes with N-GDY, along with a target-induced development of DNA nanostructure arrays. AuNPs/N-GDY serves as dual electrode substrates, and AuNPs/Mn3O4 nanozymes are surface-loaded as the bioanode. The biocathode is constructed by introducing DNA nanospace arrays onto the electrode through target-induced methods. [Ru(NH3)6]3+ is embedded into the nucleic acid double-helix scaffold via electrostatic adsorption, generating an EOCV signal that is strongly correlated with the target concentration. To further enhance sensitivity, the detection platform is combined with a capacitor to amplify the detection signal, utilizing its high power density, which results in a 22.5-fold increase in sensitivity. The method offers a linear detection range of 0.0001 to 10,000 pM and an detection limit of 32.5 aM (S/N = 3). This method supplies a novel approach for real-time monitoring and competent oversight of pokkah boeng disease pathogenic bacteria.

Exploring the Modern Fungicides for the In vitro Management of Pokkah Boeng Disease of Maize Caused by Fusarium verticillioides (SACC.) Nirenberg

Pokkah boeng disease caused by Fusarium verticillioides is an emerging fungal disease affecting maize recently leading to substantial yield losses. The increasing resistance of pathogens to older fungicides, along with environmental concerns, underscores the need for new management strategies that incorporates next-generation fungicides. In response to this challenge, a comprehensive and systematic survey was conducted in 2022-23 across the Dharwad district of Karnataka. The survey documented disease symptoms such as chlorosis at the base of leaves, malformation and twisting of the foliage. The pathogen was isolated and confirmed as F. verticillioides through molecular diagnosis. In the present study, the pathogen was tested for in vitro efficacy of modern fungicides at three different concentrations viz., 0.1, 0.15 and 0.2 per cent using poisoned food technique. In vitro testing of new generation fungicides revealed that, Tebuconazole 25.9%EC and combinations fungicides viz., Carbendazim 12%+Mancozeb 63%WP and Trifloxystrobin 25% + Tebuconazole 50%WG resulted in 100 per cent inhibition of the mycelial growth of the pathogen. Hexaconazole 5%SC, Azoxystrobin 23%SC, Trifloxystrobin 25%SC, Tricyclazole 18%+Mancozeb 62% WP and Azoxystrobin 18.2%+Difenoconazole 11.4%SC inhibited mycelial growth by 99.15, 85.49, 85.57 93.08 and 86.87 per cent, respectively. The mycelial growth in fungicide-treated media appeared either fluffy or cottony. Among the fungicides tested, Carbendazim 12% + Mancozeb 63% WP, Trifloxystrobin 25% + Tebuconazole 50% WG, and Tebuconazole 25.9%EC demonstrated 100 per cent efficacy in inhibiting pathogen mycelial growth, even at a low concentration of 0.1%. These results suggest a promising direction for disease management, while the other fungicides tested were found to be less effective.

Fusarium sacchari hypovirus 1, a Member of Hypoviridae with Virulence Attenuation Capacity in Phytopathogenic Fusarium Species.

In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.

A chromosomal-scale genome assembly of modern cultivated hybrid sugarcane provides insights into origination and evolution.

Sugarcane is a vital crop with significant economic and industrial value. However, the cultivated sugarcane's ultra-complex genome still needs to be resolved due to its high ploidy and extensive recombination between the two subgenomes. Here, we generate a chromosomal-scale, haplotype-resolved genome assembly for a hybrid sugarcane cultivar ZZ1. This assembly contains 10.4 Gb genomic sequences and 68,509 annotated genes with defined alleles in two sub-genomes distributed in 99 original and 15 recombined chromosomes. RNA-seq data analysis shows that sugar accumulation-associated gene families have been primarily expanded from the ZZSO subgenome. However, genes responding to pokkah boeng disease susceptibility have been derived dominantly from the ZZSS subgenome. The region harboring the possible smut resistance genes has expanded significantly. Among them, the expansion of WAK and FLS2 families is proposed to have occurred during the breeding of ZZ1. Our findings provide insights into the complex genome of hybrid sugarcane cultivars and pave the way for future genomics and molecular breeding studies in sugarcane.

Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

Analysis of metabolome and microbiome revealed the resistance mechanisms in sugarcane cultivars with high resistance to pokkah boeng disease

BackgroundEndophytes are reported to play a role in resistance against plant pathogens. Understanding the metabolite-mediated endophytic microbiota composition in plants provides insights to improve plant stress resistance. In this study, via metabolome and microbiome analyses, we aimed to elucidate the resistance mechanism of sugarcane cultivars with high resistance to sugarcane pokkah boeng disease (PBD). The endophytic microbial composition and metabolites in the stems of various sugarcane cultivars with high resistance (HR) or high susceptibility (HS) to PBD were analyzed.ResultsThe results revealed that the endophytic fungi with biocontrol effects such as Shinella, Dechloromonas, and Microbacter were significantly enriched, and the abundance of pathogenic fungi such as Fusarium, Ramichloridium, Scleroramularia, Phaeosphaeriopsis, Sarocladium, Zygophiala, Gibberella, Pseudocercospora, Cyphellophora, Monocillium, Apiotrichum, Microsphaeropsis, and Scleroramularia significantly reduced in the stems of HR cultivars. Additionally, six metabolites [citric acid, isocitrate, malic acid, PC(16:0/0:0), phosphocholine, and lysoPC(16:0)] were significantly related to the endophytes in the stems of HR cultivars.ConclusionsThese results suggested that more abundance of antagonistic microbes and highly active metabolic functions of endophytes in the HR cultivars were the important mechanisms underlying their higher resistance to PBD.Graphical abstract

FsCGBP, a Cutinase G-Box Binding Protein, Regulates the Growth, Development, and Virulence of Fusarium sacchari, the Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari is a causal agent of sugarcane Pokkah boeng, an important fungal disease that causes a considerable reduction in yield and sugar content in susceptible varieties of sugarcane worldwide. Despite its importance, the fungal factors that regulate the virulence of this pathogen remain largely unknown. In our previous study, mapping of an insertional mutant defect in virulence resulted in the identification of a cutinase G-box binding protein gene, designated FsCGBP, that encodes a C2H2-type transcription factor (TF). FsCGBP was shown to localize in the nuclei, and the transcript level of FsCGBP was significantly upregulated during the infection process or in response to abiotic stresses. Deletion or silencing of FsCGBP resulted in a reduction in mycelial growth, conidial production, and virulence and a delay in conidial germination in the F. sacchari. Cutinase genes FsCUT2, FsCUT3, and FsCUT4 and the mitogen-activated protein kinase (MAPK) genes FsHOG1, FsMGV1, and FsGPMK1, which were significantly downregulated in ΔFsCGBP. Except for FsHOG1, all of these genes were found to be transcriptionally activated by FsCGBP using the yeast one-hybrid system in vitro. The deletion of individual cutinase genes did not result in any of the phenotypes exhibited in the ΔFsCGBP mutant, except for cutinase activity. However, disruption of the MAPK pathway upon deletion of FsMGV1 or FsGPMK1 resulted in phenotypes similar to those of the ΔFsCGBP mutant. The above results suggest that FsCGBP functions by regulating the MAPK pathway and cutinase genes, providing new insights into the mechanism of virulence regulation in F. sacchari.

Biochar amendments improve soil functionalities, microbial community and reduce Pokkah boeng disease of sugarcane

BackgroundBiochar is mainly used to enhance soil fertility, microbial activity, improve plant performance and reduce disease incidence.MethodsA greenhouse experiment was designed to investigate the response of biochar on rhizosphere soil chemical properties, enzyme activity, microbial communities, and sugarcane Pokkah boeng disease (PB). Two sugarcane varieties Zhongzhe 9 (Z9) and ROC22, susceptible/resistant to PB, were cultivated and treated with: no biochar, 15 t ha−1 biochar, and 30 t ha−1 biochar.ResultsThe amendment of 30 t ha−1 of biochar (B2) significantly improve soil pH by 1.50% and 9.61% compared with that of B1 and B0, followed by 0.51% increase by 15 t ha−1 of biochar (B1) compared with that of control (B0). The application of 15 t ha−1 biochar significantly increased available phosphorus (AP) and ammonium nitrogen (NH4+–N) by 209.93 mg kg−1 and 12.1 mg kg−1, while the application of 30 t ha−1 of biochar significantly increased 241.04 mg kg−1 of available potassium (AK) (P < 0.05). Furthermore, biochar application increased the activities of soil acid phosphatase (S-ACP), urease (S-UE), and sucrase (S-SC). Alpha diversity analysis showed that the addition of biochar significantly altered the variety and abundance of rhizosphere microorganisms (P < 0.01) and increased the relative abundance of beneficial microorganisms Rhodanobacteraceae, Stachybotryaceae, Agaricacea, Talaromyces, Nectriaceae, Sistotrema, and Bacillus (P < 0.01). There was a significant decrease in the relative abundance of the soil pathogen Fusarium (P < 0.01).ConclusionThese findings suggested that the application of 15 t ha−1 biochar could bring desirable variations in soil functionalities, modulate soil microbial community by increasing soil health and reduce the disease index of PB.Graphical

Small secreted effector protein from Fusarium sacchari suppresses host immune response by inhibiting ScPi21-induced cell death.

Fusarium sacchari is one of the primary pathogens causing pokkah boeng disease, which impairs the yield and quality of sugarcane around the world. Understanding the molecular mechanisms of the F. sacchari effectors that regulate plant immunity is of great importance for the development of novel strategies for the persistent control of pokkah boeng disease. In a previous study, Fs00367 was identified to inhibit BAX-induced cell death. In this study, Fs00367nsp (without signal peptide) was found to suppress BAX-induced cell death, reactive oxygen species bursts and callose accumulation. The amino acid region 113-142 of Fs00367nsp is the functional region. Gene mutagenesis indicated that Fs00367 is important for the full virulence of F. sacchari. Ayeast two-hybrid assay revealed an interaction between Fs00367nsp and sugarcane ScPi21 in yeast that was further confirmed using bimolecular fluorescence complementation, pull-down assay and co-immunoprecipitation. ScPi21 can induce plant immunity, but this effect could be blunted by Fs00367nsp. These results suggest that Fs00367 is a core pathogenicity factor that suppresses plant immunity through inhibiting ScPi21-induced cell death. The findings of this study provide new insights into the molecular mechanisms of effectors in regulating plant immunity.

Identification of common fungal extracellular membrane (CFEM) proteins in Fusarium sacchari that inhibit plant immunity and contribute to virulence

ABSTRACT Fusarium sacchari is one of the primary pathogens causing pokkah boeng disease in sugarcane in China. Common fungal extracellular membrane (CFEM) domain-containing proteins only exist in fungi and play a significant role in the interaction of pathogens and plants. However, the CFEM proteins in F. sacchari were not identified and functionally resolved. Of the 20 FsCFEM proteins identified from the genome of F. sacchari , 16 FsCFEM genes were cloned, while 4 FsCFEM proteins (Fs06761, Fs08184, Fs10706, and Fs13617) were identified as effector proteins that could suppress the programmed cell death triggered by BCL2-associated X protein in Nicotiana benthamiana . Subcellular localization analysis revealed that these four effectors could enter plant cells and are mainly located in the cell membrane and nucleus. By the yeast YTK12 secretion system, the signal peptides of four CFEM proteins were functionally verified. The expression levels of these four effector proteins were significantly increased in the F. sacchari -infected sugarcane plants. Through fungal gene knockout, three genes (Fs06761, Fs08184, and Fs13617) were found to be important pathogenic factors for pathogens. Our results support that the FsCFEM effector proteins were virulence effectors and might be involved in regulating plant immunity. IMPORTANCE Common fungal extracellular membrane (CFEM) domain-containing protein has long been considered an essential effector, playing a crucial role in the interaction of pathogens and plant. Strategies aimed at understanding the pathogenicity mechanism of F. sacchari are eagerly anticipated to ultimately end the spread of pokkah boeng disease. Twenty FsCFEM proteins in the genome of F. sacchari have been identified, and four FsCFEM effector proteins have been found to suppress BCL2-associated X protein-triggered programmed cell death in N. benthamiana . These four effector proteins have the ability to enter plant cells and inhibit plant immunity. Furthermore, the expression of these four FsCFEM effector proteins significantly increases during the infection stage, with the three of them playing an essential role in achieving full virulence. These study findings provide a direction toward further exploration of the immune response in sugarcane. By applying these discoveries, we can potentially control the spread of disease through techniques such as host-induced gene silencing.

Evaluation of Sugarcane varieties against Pokkah boeng disease, role of meteorological factors and its management

Sugarcane (Saccharum officinarum L.) is widely grown as cash crop in India and provides employment to over a million of people directly or indirectly. Recently, increasing trends of Pokkah boeng disease caused by (Fusarium proliferatum) incidence were noticed in most of the sugarcane varieties grown in Bihar which ranged between 5-15%. A field trial was conducted during 2016-2018 at sugarcane research Institute farm, RPCAU, Pusa. In this study, thirty one sugarcane varieties were evaluated against Pokkah boeng disease, out of thirty one sugarcane varieties evaluated against Pokkah boeng disease, sixteen varieties showed resistant, ten varieties showed moderately susceptible while, four varieties were graded as susceptible to Pokkah boeng disease. The correlation analysis between meteorological factors and disease incidence signify that minimum temprature showed significant positive correlation whereas, relative humidity of 07 hrs, 14 hrs. and rainfall showed highly positive correlation while, sunshine displayed significant but statistically was negative relationship with Pokkah boeng disease incidence. Sett treatment with fungicide Carbendazim @ 0.1% and three foliar sprays with Carbendazim @ 0.1% at 15 days intervals revealed that highest germination and maximum control of disease incidence in all the tested varieties.

Expert system for smart farming for diagnosis of sugarcane diseases using machine learning

Agriculture is one of the oldest occupations in the world and continues to exist today. In some form or another, the world’s population depends on agriculture for its needs. The major loss in sugarcane production in India is due to pests, plant disease, malnutrition, and nutrient deficiency in plants. To identify these diseases, farmers go to local farmers, experts, agricultural people, and fellow neighbors to identify the problem caused. In some cases, their information may be adequate, but in others it is not. These people cannot solve all the problems caused by their crops can be solved by these people; there is a need to accurately predict the correct disease and provide the proper treatment at the right time. This can only be done by applying machine learning-based Internet of Things solutions in real time. This article proposes a method for a smart farming system to address the needs of farmers producing sugarcane in India by applying intelligent solutions that use image processing and soft computing. Four sugarcane diseases are investigated, such as Eyespot, Leaf Scald, Yellow Leaf, and Pokkah Boeng, and three characteristics such as color, shape, and texture. Images were used for training data in Artificial Neural Network (ANN), Neuro-Fuzzy, and Case-Based Reasoning (CBR) algorithms, and the performance of the feature extraction technique was evaluated in terms of sensitivity, specificity, F1 score, and accuracy.

Pectate Lyase from Fusarium sacchari Induces Plant Immune Responses and Contributes to Virulence.

Fusarium sacchari is one of the primary pathogens causing Pokkah Boeng disease (PBD) in sugarcane in China. Pectate lyases (PL), which play a critical role in pectin degradation and fungal virulence, have been extensively studied in major bacterial and fungal pathogens of a wide range of plant species. However, only a few PLs have been functionally investigated. In this study, we analyzed the function of the pectate lyase gene, FsPL, from F. sacchari. FsPL is a key virulence factor of F. sacchari and can induce plant cell death. FsPL also triggers the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) response in Nicotiana benthamiana, as reflected by increases in reactive oxygen species (ROS) production, electrolyte leakage, and callose accumulation, as well as the upregulation of defense response genes. In addition, our study also found that the signal peptide of FsPL was necessary for induced cell death and PTI responses. Virus-induced gene silencing showed that FsPL-induced cell death in Nicotiana benthamiana was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Thus, FsPL may not only be a critical virulence factor for F. sacchari but may also induce plant defense responses. These findings provide new insights into the functions of pectate lyase in host-pathogen interactions. IMPORTANCE Pokkah Boeng disease (PBD) is one of the main diseases affecting sugarcane in China, seriously damaging sugarcane production and economic development. Therefore, it is important to clarify the pathogenic mechanisms of this disease and to provide a theoretical basis for the breeding of PBD-resistant sugarcane strains. The present study aimed to analyze the function of FsPL, a recently identified pectate lyase gene from F. sacchari. FsPL is a key virulence factor of F. sacchari that induces plant cell death. Our results provide new insights into the function of pectate lyase in host-pathogen interactions.

Genetic Diversity and Pathogenicity of Fusarium fujikuroi Species Complex (FFSC) Causing Sugarcane Pokkah Boeng Disease (PBD) in China.

Pokkah boeng disease (PBD), a sugarcane foliar disease, is caused by various Fusarium spp. within the Fusarium fujikuroi species complex (FFSC). In the current study, we investigated the diversity of Fusarium spp. associated with PBD in China. In total, 320 leaf samples displaying PBD symptoms were collected over 10 consecutive years (2012 to 2021), during winter and summer, from six various sugarcane-growing regions (Guangxi, Yunnan, Guangdong, Zhejiang, Hainan, and Fujian) in China. Phylogenetic analysis of Fusarium spp. was reconstructed using translation elongation factor 1-α, and DNA-directed RNA polymerase II largest subunit and second-largest subunit multigene sequences. Evolutionary studies of these regions categorized the isolates into four FFSC species (F. sacchari, F. proliferatum, F. verticillioides, and F. andiyazi). The identified isolates, which developed irregular necrotic patches and rotting symptoms on the sugarcane plant after approximately 30 days were tested for their pathogenicity. Symptoms that appeared during pathogenicity testing were consistent with those observed under field conditions. Each strain of the pathogenic Fusarium spp. belonged to different vegetative compatibility groups (VCGs), and there was no affinity between VCGs. Our results contribute to understanding FFSC and accurately identifying Fusarium spp. associated with the sugarcane crop.

Occurrence and field evaluation of sugarcane genotypes for resistance to pokkah boeng disease in China

AbstractSugarcane pokkah boeng disease (PBD), caused by aFusariumspecies complex, is one of the most critical diseases causing a significant economic loss of sugarcane production in China. It is important to standardize the evaluation method for screening sugarcane cultivars for resistance to PBD. Field trials were conducted to investigate the occurrence of sugarcane PBD in different months and growth stages from 2018 to 2019 and to evaluate the PBD resistance of 169 sugarcane genotypes from 2019 to 2022. PBD mainly occurred in the early and middle elongation stages at a plant height of approximately 1–2 m. The most severe and common incidence of PBD was from June to August. Variance analysis showed genotype, year and the interaction of genotype and year had a significant effect on disease incidence and disease index. The 169 sugarcane genotypes were divided into five categories by cluster analysis and discriminant analysis, of which 33 were rated as highly resistant, 37 as resistant, 38 as moderately susceptible, 26 as susceptible and 35 as highly susceptible. These findings enhance our understanding of sugarcane resistance to PBD and lay the foundation for breeding new resistant varieties.

First report of Fusarium verticillioides causing Pokkah boeng disease on maize in India.

Maize is a widely grown cereal crop in India and ranks third to wheat and rice in production (https://iimr.icar.gov.in). During a field survey in Kharif season in 2018, foliar chlorosis at the base and middle of leaves, and twisted top symptoms were observed in 40-50 days old maize plants in Belagavi district, Karnataka, India. Again during Kharif season in 2021, similar symptoms were observed on commercial maize hybrids and sugarcane at Agricultural Research Station, Sankeshwar Karnataka. The symptoms resembled Pokkah boeng disease of sugarcane (Vishwakarma et al. 2013). Symptomatic sugarcane and maize leaves were sampled, surface sterilized with 1.0% sodium hypochlorite, and 70% ethanol, and transferred on Potato dextrose agar, incubated for 10 days at 27±1°C. Fungal growth initiated with white mycelium later turned to pinkish-white with hyaline spores. The morphological features and sporulation patterns of maize and sugarcane samples were similar (e-Xtra 1). Microconidia were formed in long chains and clusters with oval to club-shaped, 0-septate, monophialide-borne microspores. DNA from representative pure culture isolates was extracted using the CTAB protocol (Doyle and Doyle, 1990). The ITS region of r-DNA was amplified with ITS1/ITS4 primers and sequenced. BLAST analyses of sequences of maize and sugarcane culture isolates at NCBI database revealed 100% homology with Fusarium verticillioides MK264336 (Lin et al., 2016). PCR amplification with Fusarium verticilliodes specific primers VER1/VER2 (Mule et al., 2004) confirmed the organism. CBS-KNAW Fungal Biodiversity Centre's Fusarium MLST database also revealed over 98.89% homology with Fusarium verticilliodes (NRRL 46612). The fungal isolates were named Fusarium verticilliodes maize isolate SNK 01 (ON110289) and Fusarium verticilliodes sugarcane isolate SNK 01 (ON564879), and their sequences were deposited in the GenBank. To test pathogenicity, artificial inoculation using maize isolate SNK 01 and cross-inoculation of sugarcane isolate SNK 01 were done on ten maize plants by spraying a conidial suspension (2×106 conidia ml-1) on nonwounded leaves. The plants sprayed with sterile water were used as control. After ten days, typical Pokkah boeng symptoms were observed in the plants inoculated with both maize and sugarcane isolates. Diseased leaves turned pale yellowish-green with small brown spots and a chlorotic appearance, further, these developed into stripes (e-Xtra 2). Wrinkling of leaves was noticed followed by splitting and rotting. No symptoms were noticed in the water-treated control. The pathogens re-isolated from diseased plants inoculated with maize and sugarcane isolates were similar morphologically and identical to the original isolates, fulfilling Koch's postulates. Hitherto, Fusarium verticilliodes was known to cause post-flowering stalk rot in maize. However, this is the first report of Pokkah boeng disease on maize in India caused by F. verticillioides. Considering the economic value of the maize crop, this identification can help develop appropriate disease management strategies to control the disease. References Lee, S. B., et al. 1988. A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet. Newsl. 35:23-24. Lin, Z., et al. 2016. Deciphering transcriptomic response of Fusarium verticillioides in relation to nitrogen availability and the development of sugarcane Pokkah boeng disease. Sci. Rep. 6, 29692. Mule, G., et al. 2004. A Species-Specific PCR assay based on the Calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. European J. Plant Path. 110:495-502 Vishwakarma, S.K., et al. 2013. Pokkah Boeng: an emerging disease of sugarcane. J. Plant Pathol. Microb. 4(3):170. https://iimr.icar.gov.in. Director's desk, ICAR-Indian Institute of Maize Research. (Accessed September 8, 2022).