Plant Assays Research Articles

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Biocontrol potential of lipopeptides produced by the novel Bacillus altitudinis strain TM22A against postharvest Alternaria rot of tomato

Bacillus genus exhibits antagonistic potential against a broad range of pathogenic fungi. This study presents the antifungal potential of lipopeptides (LPs) produced by a novel strain Bacillus altitudinis TM22A to suppress the infection of Alternaria rot on tomato. We investigated the antifungal effect of ten Bacillus species against Alternaria alternata, where four Bacillus strains TM22A, MCM61, S2, and CPCF54 exhibited antifungal activity. The LPs extracted from TM22A, MCM61, S2, and CPCF54 restricted mycelial growth and conidial germination of A. alternata in vitro with the highest inhibition produced by TM22A. Liquid chromatography and mass spectrometry (LC-MS) analysis revealed the presence of surfactin, fengycin, iturin, bacillibactin, and bacilysin in strain TM22A at 1036.69, 1435.76, 1085.58, 883.26, and 271.13 m/z respectively. In an In planta assay LPs from TM22A caused the lowest lesion diameter and disease incidence on tomato fruit. Furthermore, the activity of defense enzymes viz, catalase (CAT), polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL) was enhanced in Bacillus LPs treated tomato fruit with the more pronounced impact by TM22A. Postharvest quality analysis revealed little impact on ascorbic acid, fruit pH, total soluble solids (TSS), titratable acidity (TA), weight loss, and fruit firmness compared to healthy control. This is the first comprehensive study to reveal the antifungal effect of Bacillus-derived LPs against postharvest Alternaria rot of tomato and its role in defense elicitation to protect the fruit from pathogen invasion.

A host‐derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

AbstractIntroductionThe application of host‐derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for the treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such as HLB‐causing Candidatus liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defence responses. Also, chimeric peptides can have an off‐target impact on the structure and function of plant‐associated microbiomes. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titre. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titre, inducing plant defence response and impacting the microbiome associated with the citrus phyllosphere.Material and MethodLeaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected without symptoms). In planta assay was performed by dipping the leaves in the 0, 5 and 25 μM of UGK17 solutions for 48 h. The quantifications of CLas titre and pathogenesis‐related (PR) gene expression were done by quantitative polymerase chain reaction (qPCR) and reverse transcription‐qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structures. In addition, we performed an ex planta assay to assess the effect of UGK17 on the growth of bacterial isolates including Liberibacter crescens instead of unculturable CLas predominant in the phyllosphere.ResultThe UGK17 treatment reduced the CLas titre in both asymptomatic and symptomatic citrus leaves, regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. In ex planta assay, the growth of L. crescens along with four other strains belonging to the family Rhizobiaceae, was significantly inhibited by the UGK17 while the growth of 74 strains were unaffected. Additionally, there was no statistically significant changes in the microbial community structure with UGK17 treatment.ConclusionOur results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting mainly Gram‐negative pathogens and enhancing the plant immune responses without impairing the indigenous microbial community in the citrus phyllosphere.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0-24h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-( +)-linalool and (E)-(-)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-( +)-linalool, a monoterpene resin volatile with a known role in plant defense.

Photorhabdus-Derived Secondary Metabolites Reduce Root Infection by Meloidogyne incognita in Cowpea.

Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from Photorhabdus bacteria, trans-cinnamic acid (t-CA), and (4E)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN Meloidogyne incognita in a cowpea. Factorial combinations of five t-CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 μg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the t-CA + PPA mixture significantly reduced the penetration of M. incognita infective juveniles (J2s) into the cowpea roots. The potential toxicity of t-CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of t-CA + PPA × nematode inoculation interactions and the t-CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll b content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of t-CA and PPA reduces M. incognita J2's ability to infect the roots without impairing plant growth or chlorophyll content.

Uncovering Genomic Features and Biosynthetic Gene Clusters in Endophytic Bacteria from Roots of the Medicinal Plant Alkanna tinctoria Tausch as a Strategy To Identify Novel Biocontrol Bacteria.

The world's population is increasing at a rate not seen in the past. Agriculture, providing food for this increasing population, is reaching its boundaries of space and natural resources. In addition, changing legislation and increased ecological awareness are forcing agriculture to reduce its environmental impact. This entails the replacement of agrochemicals with nature-based solutions. In this regard, the search for effective biocontrol agents that protect crops from pathogens is in the spotlight. In this study, we have investigated the biocontrol activity of endophytic bacteria isolated from the medicinal plant Alkanna tinctoria Tausch. To do so, an extensive collection of bacterial strains was initially genome sequenced and in silico screened for features related to plant stimulation and biocontrol. Based on this information, a selection of bacteria was tested in vitro for antifungal activity using direct antagonism in a plate assay and in planta with a detached-leaf assay. Bacterial strains were tested individually and in combinations to assess the best-performing treatments. The results revealed that many bacteria could produce metabolites that efficiently inhibit the proliferation of several fungi, especially Fusarium graminearum. Among these, Pseudomonas sp. strain R-71838 showed a strong antifungal effect, in both dual-culture and in planta assays, making it the most promising candidate for biocontrol application. Using microbes from medicinal plants, this study highlights the opportunities of using genomic information to speed up the screening of a taxonomically diverse set of bacteria with biocontrol properties. IMPORTANCE Phytopathogenic fungi are a major threat to global food production. The most common management practice to prevent plant infections involves the intensive use of fungicides. However, with the growing awareness of the ecological and human impacts of chemicals, there is a need for alternative strategies, such as the use of bacterial biocontrol agents. Limitations in the design of bacterial biocontrol included the need for labor-intensive and time-consuming experiments to test a wide diversity of strains and the lack of reproducibility of their activity against pathogens. Here, we show that genomic information is an effective tool to select bacteria of interest quickly. Also, we highlight that the strain Pseudomonas sp. R-71838 produced a reproducible antifungal effect both in vitro and in planta. These findings build a foundation for designing a biocontrol strategy based on Pseudomonas sp. R-71838.

Suppression of tomato bacterial speck disease (Pseudomonas syringae pv. tomato (Okabe) Young, Dye, & Wilkie) via induced systemic resistance by Pseudomonas and Bacillus strains

Bacterial speck Pseudomonas syringae pv. tomato (Pst) (Okabe) Young, Dye, & Wilkie is a widespread disease in tomato plants. Four plant growth-promoting rhizobacterial (PGPR) strains 5(3), 68(2), 36(1), and 47(3) played a significant role (50% and higher) in reducing spot disease severity. Selected strains were identified as Pseudomonas koreensis 5(3), Bacillus mycoides 68(2), Bacillus mojavensis 36(1), and Bacillus simplex 47(3) using the MALDI Biotyper classification system. In planta assay using tomato seedlings were inoculated with the bacterial strains alone or in dual combination. Pseudomonas koreensis 5(3) (51.9%–74.29%) and Bacillus mycoides 68(2) (36.70%–65.56%) both provided a significant reduction in foliar severity caused by bacterial speck disease agent Pseudomonas syringae pv. tomato (Okabe) Young, Dye, & Wilkie. Bacillus simplex 47(3) and Bacillus mojavensis 36(1) were successful only in combined treatments. Defense enzymes Proline, Peroxidase, and Catalase were induced by PGPR strains in comparison with those of control plants. Hydrogen peroxide (H2O2) and callose deposition were evident at reaction sites induced by PGPR strains. The accumulation of callose, H2O2, and high levels of defense enzymes via the treatment of PGPRs might play a significant role in a practical, safe, and effective way to control Pseudomonas syringae pv. tomato.

Assessment of Tunisian Trichoderma Isolates on Wheat Seed Germination, Seedling Growth and Fusarium Seedling Blight Suppression.

Beneficial microorganisms, including members of the Trichoderma genus, are known for their ability to promote plant growth and disease resistance, as well as being alternatives to synthetic inputs in agriculture. In this study, 111 Trichoderma strains were isolated from the rhizospheric soil of Florence Aurore, an ancient wheat variety that was cultivated in an organic farming system in Tunisia. A preliminary ITS analysis allowed us to cluster these 111 isolates into three main groups, T. harzianum (74 isolates), T. lixii (16 isolates) and T. sp. (21 isolates), represented by six different species. Their multi-locus analysis (tef1, translation elongation factor 1; rpb2, RNA polymerase B) identified three T. afroharzianum, one T. lixii, one T. atrobrunneum and one T. lentinulae species. These six new strains were selected to determine their suitability as plant growth promoters (PGP) and biocontrol agents (BCA) against Fusarium seedling blight disease (FSB) in wheat caused by Fusarium culmorum. All of the strains exhibited PGP abilities correlated to ammonia and indole-like compound production. In terms of biocontrol activity, all of the strains inhibited the development of F. culmorum in vitro, which is linked to the production of lytic enzymes, as well as diffusible and volatile organic compounds. An in planta assay was carried out on the seeds of a Tunisian modern wheat variety (Khiar) by coating them with Trichoderma. A significant increase in biomass was observed, which is associated with increased chlorophyll and nitrogen. An FSB bioprotective effect was confirmed for all strains (with Th01 being the most effective) by suppressing morbid symptoms in germinated seeds and seedlings, as well as by limiting F. culmorum aggressiveness on overall plant growth. Plant transcriptome analysis revealed that the isolates triggered several SA- and JA-dependent defense-encoding genes involved in F. culmorum resistance in the roots and leaves of three-week-old seedlings. This finding makes these strains very promising in promoting growth and controlling FSB disease in modern wheat varieties.

Phytochemical Extracts of Dittrichia viscosa (L.) Greuter from Agroecological Systems: Seed Antigerminative Properties and Effectiveness in Counteracting Alternaria Leaf Spot Disease on Baby-Leaf Spinach.

Dittrichia viscosa (L.) Greuter subsp. viscosa (Asteraceae) is a perennial species naturally distributed in arid and marginal areas whose agroecological cultivation could be a useful innovation to produce quality biomass to extract phenolic-rich phytochemical blends. Here, biomass-yield trends were profiled at different growth stages under direct cropping, and inflorescences, leaves, and stems were submitted to water extraction and hydrodistillation. Then, four extracts were investigated for their biological activities in invitro and in planta assays. Extracts inhibited cress (Lepidium sativum)- and radish (Raphanus sativus)-seed germination and root elongation. All samples showed dose-dependent antifungal activity in the plate experiments, inhibiting up to 65% of the growth of the fungal pathogen Alternaria alternata, a leaf-spot disease agent of baby spinach (Spinacea oleracea). However, only the extracts from dried green parts and fresh inflorescences at the highest concentration significantly reduced (54%) the extent of Alternaria necrosis on baby spinach. UHPLC-HRMS/MS analysis revealed that the main specialized metabolites of the extracts are caffeoyl quinic acids, methoxylated flavonoids, sesquiterpene compounds such as tomentosin, and dicarboxylic acids, which may explain the observed bioactivity. Plant extracts obtained through sustainable methodology can be effective in biological agricultural applications.

Colonization and endophytic effect of Beauveria bassiana (Bals.-Criv.) Vuill. UHSB-END1 against Myzus persicae (Sulzer) and Plutella xylostella (L.) in cabbage

BackgroundFungal microbial biocontrol agent playing an important role in sustainable pest management with low cost. Conidial contact with cuticle is sufficient to cause disease in insects by entomopathogenic fungi (EPF) compared to bacteria and viruses which required ingestion of contaminated food. The field level application of fungi resulted inconsistent in their efficacy against insects as they withstand in the agroecosystem with adverse environmental conditions. To overcome this bottleneck, endophytic EPF, Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae: Hypocreales), is well-studied for the management of insects of cultivated crops and got promising results. The colonization of endophytic isolates varied from one host plant to another and virulence against insects too. Hence, the study was undertaken to know the colonization of indigenous B. bassiana UHSB-END1 isolate in cabbage plant and its efficacy against major insects of cabbage.ResultsThe indigenous isolate of B. bassiana UHSB-END1 was able to colonize cabbage in all the methods of inoculations (seed treatment, seedling root dip, soil drenching, foliar spray and combination treatment) at 30, 45 and 60 days of post-inoculation (dpi). However, the colonization of the fungus inside the cabbage was restricted to tissues of inoculation, and movement from the site of inoculation to other parts of the plant was poor. In the present study, colonization was the highest at 30 dpi in all methods of colonization with restricted colonization to the site of exposure of fungus. Further, in vivo and in planta assay confirmed the effectiveness of B. bassiana UHSB-END1 colonized cabbage against Myzus persicae (Sulzer) and Plutella xylostella (Linnaeus). Under in vivo conditions the maximum mortality of M. persicae and P. xylostella was recorded at 30 dpi. It was decreased slightly at 45 dpi, and the lowest morality was recorded at 60 dpi. In planta experiment also proved better efficacy against both the test insects. Wherein mortality of M. persicae ranged from 22 to 36% five days after release (DAR), it was increased to 48–68% at 10 DAR and reached highest mortality rate at 15DAR (72–96%). Similarly, mortality of P. xylostella ranged from 14 to 24% after five DAR, and it was 27–44% at 10 DAR, and maximum mortality rate of larvae were recorded at 15 DAR (64–96%).ConclusionThe present study reported 100% colonization of cabbage plant by indigenous B. bassiana UHSB-END1 at 30 dpi in combination of treatment and showed a maximum efficacy in managing the population of major insects of cabbage. Introducing this indigenous isolate will pave a ray of hope in managing the both key insects (P. xylostella and M. persicae) in cabbage agroecosystem without disturbing the environment and further helps in production of cabbage with minimum pesticide residue for consumers.

Biocontrol Potential of Some Rhizospheric Soil Bacterial Strains against Fusarium culmorum and Subsequent Effect on Growth of Two Tunisian Wheat Cultivars

PGPR (Plant Growth Promoting Rhizobacteria) are used as biofertilizers and biological control agents against fungi. The objective of this work was to evaluate the antagonistic activities of some bacterial strains isolated from soil against four phytopathogenic fungal strains (Fusarium graminearum, F. culmorum, Phytophthora sp. and Verticillium dahlia). Two strains having an antagonist effect on fungi and displaying the maximum of plant growth promoting (PGP) traits were selected for further study and identified as Bacillus subtilis and B. amyloliquefaciens respectively. In planta assays demonstrated that the two Bacillus strains are able to enhance plant growth of two wheat cultivars in absence of nitrogen and protect them against F. culmorum. Pot experiments performed in a greenhouse showed that wheat plants inoculation with two bacterial strains reduce F. culmorum disease severity correlated with the accumulation of phenolic compounds and chlorophyll content. These could partly explain the effectiveness of these bacteria in protecting Tunisian durum wheat cultivars against F. culmorum. Application B. amyloliquefaciens, showed better protection than B. subtilis although the last one enhanced more the plant growth of two wheat cultivars in absence of fungus. Hence, combination of two bacterial strains could be a strategic approach to enhance plant growth and control plant diseases.

Monitoring for a new I3 resistance gene-breaking race of F. oxysporum f. sp. lycopersici (Fusarium wilt) in California processing tomatoes following recent widespread adoption of resistant (F3) cultivars: Challenges with race 3 and 4 differentiation methods.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Fol), causes losses in tomato production worldwide, with major impacts on Californian tomato processing. Single-gene resistance is the primary management tool, but its efficacy has been compromised following the emergence of two successive resistance-breaking races, which, in California, emerged within 12years of resistance deployment. Fol race 3-resistant (F3) processing tomato cultivars (containing the I3 resistance gene) were deployed in the state starting in approximately 2009. The emergence of a new resistance-breaking race (which would be called race 4) is imminent, and early detection will be critical to delay the spread while new resistance is sought. The detection of Fol race 4 is challenged by the lack of validated, rapid, and accurate diagnostic tools. In evaluating in planta phenotyping methods, this study found that rapid seedling phenotyping is not reliable and generates false positives for nonpathogens. Longer (10weeks) mature plant assays are the most reliable, but may not be sufficiently timely. As an additional challenge, based on field and greenhouse studies, Fol race 3 can cause symptoms in resistant F3 cultivars at frequencies greater (30%) than expected for off-types (<2%). We developed a three-F3 cultivar in planta assay to overcome the challenges this posed to differentiating Fol race 3 and Fol race 4. Using the assay, we determined that all putative resistance-breaking cases were Fol race 3; Fol race 4 was not detected in these early survey efforts. These results highlight the need for developing rapid Fol race 4 detection tools and a better understanding of the factors underlying inconsistent I3 gene expression in Fol race 3.

Sphingolipid Long-Chain Base Signaling in Compatible and Non-Compatible Plant–Pathogen Interactions in Arabidopsis

The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB- MPK6- ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.

Evaluation of the mechanism of action of Bacillus spp. to manage Meloidogyne incognita with split root assay, RT-qPCR and qPCR.

The goal of this research is to determine the mechanism of action of two Bacillus spp. that can manage Meloidogyne incognita population density in cotton. The overall objectives are 1) determine the efficacy and direct antagonistic capabilities of the Bacillus spp. and 2) determine the systemic capabilities of the Bacillus spp. The greenhouse in planta assay indicated B. amyloliquefaciens QST713 and B. firmus I-1582 could manage M. incognita similarly to the chemical standard fluopyram. An in vitro assay determined that B. firmus I-1582 and its extracted metabolites were able to directly manage M. incognita second stage juveniles by increasing mortality rate above 75%. A split root assay, used to determine systemic capabilities of the bacteria, indicated B. amyloliquefaciens QST713 and B. firmus I-1582 could indirectly decrease the nematode population density. Another species, B. mojavensis strain 2, also demonstrated systemic capabilities but was not a successful biological control agent because it supported a high population density in greenhouse in planta assay and in the split root assay. A RT-qPCR assay was used to confirm any systemic activity observed in the split root assay. At 24 hours both B. amyloliquefaciens QST713 and B. firmus I-1582 upregulated one gene involved in the initial stages of JA synthesis pathway but not another gene involved in the later stages of JA synthesis. These results point to a JA intermediate molecule, most likely OPDA, stimulated by the bacteria rather than JA in a short-term systemic response. After 1 week, the Bacillus spp. stimulated a SA-responsive defense related gene. The long-term systemic response to the Bacillus spp. indicates salicylic acid also plays a role in defense conferred by these bacteria. The final assay was a qPCR to determine the concentration of the bacteria on the cotton roots after 24 days. Bacillus amyloliquefaciens QST713 and B. firmus I-43 1582 were able to colonize the root successfully, with the concentration after 24 days not significantly differing from the concentration at inoculation. This study identifies two bacteria that work via systemic resistance and will help aid in implementing these species in an integrated management system.

Bacillus velezensis (strains A6 & P42) as a potential biocontrol agent against Klebsiella variicola, a new causal agent of soft rot disease in carrot.

Bacterial soft rot is one of the most devastating diseases and a major constraint encountered during carrot farming. Biological agents are the best eco-friendly alternatives to agrochemicals to manage soft rot disease to ensure environmental sustainability. In this study, about eight isolates of bacterial pathogen causing soft rot in carrots were collected from Karnataka, India. Based on the 16S rRNA sequencing the pathogen isolates causing soft rot of carrot were identified as Klebsiella variicola. The morphological characteristics of K. variicola was investigated under scanning electron microscopy. The pathogenicity assay showed that all eight isolates were pathogenic to the carrot. An in vitro and in planta assay of two novel strains of Bacillus velezensis (A6 and P42) against K. variicola indicated that both strains had strong antagonistic activity against all the pathogen strains. Furthermore, the volatile bioactive compounds produced by A6 and P42 strains were analyzed in GC-MS, which revealed the presence of 10 and 6 bioactive compounds in their culture filtrate, respectively, with antibacterial and antifungal properties. The present study suggests that both A6 and P42 strains of B. velezensis were antagonistic to K. variicola and can be used as biocontrol agents to manage soft rot diseases of carrot under field conditions.

Native endophytes from maize as potential biocontrol agents against bacterial top rot caused by cross-kingdom pathogen Klebsiella pneumoniae

Maize crop has been suffering huge economic loss due to bacterial top rot disease caused by emerging plant pathogen Klebsiella pneumoniae, which is most concerning and highly contagious to both humans and animals. At present, chemical control is considered as an important disease management approach, however biological control through native endophytes has not been reported yet. Here, initially we isolated 120 native endophytes from maize plants, and screened 12 endophytes with potential antagonistic effect in coculture assay against maize bacterial top rot pathogen. Based on strong antibacterial activity, three endophytes (MZ-4, YN-23, and F14) were selected to evaluate their plant growth-promoting traits and biocontrol efficiency through in-vitro and in-vivo antagonistic assays. The three strains could produce IAA with a maximum yield of 2.54 µg/ml, 2.96 µg/ml, 2.07 µg/ml, respectively and also inhibit the growth of 10 common plant pathogenic bacteria. In planta assay suggested that three endophytes enhanced maize growth and significantly suppressed the incidence of K. pneumoniae with control effect of about 81.2 %, 74.7 %, and 66.7 %, respectively. Lastly, selected isolates MZ-4, YN-23, and F14 were molecularly characterized and identified as Bacillus velezensis. To our knowledge, this is the first study reporting the application of maize endophyte B. velezensis as a biocontrol agent against emerging pathogen of maize bacterial top rot disease.

Expression of SIX1b and SIX1c effector genes and banana resistance genes during Foc TR4 infection on banana cultivars

Abstract. Ulilalbab AR, Widinugraheni S, Masanto, Subandiyah S, Wibowo A. 2022. Expression of SIX1b and SIX1c effector genes and banana resistance genes during Foc TR4 infection on banana cultivars. Biodiversitas 23: 5314-5322. The relative expression SIX1 genes isolates of Foc TR4 and banana resistance genes have not been intensively studied. This study aimed to determine the expression of SIX1 genes of Foc TR4 and banana resistance genes. Twelve Foc TR4 isolates were collected from several regions in Indonesia. DSI analysis was performed to categorize their virulence levels. The representative Foc TR4 isolates from different virulence levels were artificially inoculated on the Musa acuminata Colla Cavendish subgroup and cv. Barangan and incubated for 48, 72, and 96 HPI. The expressed genes were quantitatively analyzed using the qPCR technique using primers to amplify SIX1b, SIX1c, chitinase, and PR-protein 1 genes. The results categorized the virulence of Foc TR4 isolates into moderate, virulent, and high virulent. The isolates of KJG and Batu-4 were selected for gene expression study representing moderate and high virulent groups, respectively. The results of in planta assay found that the expression of SIX1b, SIX1c, chitinase, and PR-protein1 was upregulated on the inoculated plants during the incubation period. However, the expression of these genes was increasingly upregulated in both bananas at early-stage inoculation. We assumed that plant defense genes of bananas might actively encounter the common virulence mechanisms of Foc TR4 at the initial stage of inoculation.

Evaluation of commonly used nematicides with combined in in vitro and in planta bioassays

Summary For efficient use of available synthesised nematicides, bioassay-guided analysis is required to understand the behaviours of nematicides. In this study, the key nematicides on the market, including avermectin B1a, avermectin B2a, emamectin benzoate, fluensulfone, fluopyram and fosthiazate, were evaluated by both in vitro and in planta approaches with optimised solvent and nematode inoculum. We found that acetone was the only one of the six solvents tested safe for both root-knot nematode (RKN) second-stage juveniles (J2) and cucumber at the tested concentration. The optimal numbers of RKN J2 for inoculation on cucumber seedlings in the glass-tube assay, cup assay and microplot experiment were 200, 700 and 1250, respectively. No obvious phytotoxicity symptoms were observed in the in planta assays for all nematicides selected at the tested concentration in the soil drench application. When the substrate changed from pure sand to matrix-sand, the EC50 and EC90 of all six nematicides increased. Among them, fluopyram rose the most. In the microplot experiment, avermectin B2a showed the lowest EC50 but the EC90 of avermectin B2a, fluensulfone, fluopyram and fosthiazate were similar. The results are useful as a guide to the application of commonly used nematicides in the field.

Biosynthesized silver nanoparticles using Polygonatum geminiflorum efficiently control fusarium wilt disease of tomato.

Nanomaterials are gaining tremendous potential as emerging antimicrobials in the quest to find resistance-free alternatives of chemical pesticides. In this study, stable silver nanoparticles were synthesized using the aqueous extract of medicinal plant species Polygonatum geminiflorum , and their morphological features were evaluated by transmission electron microscopy, X-ray diffraction spectroscopy and energy dispersive X-ray analysis. In vitro Antifungal activity of the synthesized silver nanoparticles (AgNPs) and P. geminiflorum extract (PE) either alone or in combination (PE-AgNPs) against Fusarium oxysporum was evaluated using disc-diffusion and well-diffusion methods. In planta assay of the same treatments against Fusarium wilt diseases of tomato was evaluated by foliar spray method. Moreover, plant extract was evaluated for the quantitative investigation of antioxidant activity, phenolics and flavonoids by spectroscopic and HPLC techniques. Phytochemical analysis indicated the presence of total phenolic and flavonoid contents as 48.32 mg ± 1.54 mg GAE/g and 57.08 mg ± 1.36 mg QE/g, respectively. The DPPH radical scavenging of leaf extract was found to be 88.23% ± 0.87%. Besides, the HPLC phenolic profile showed the presence of 15 bioactive phenolic compounds. Characterization of nanoparticles revealed the size ranging from 8 nm to 34 nm with average crystallite size of 27 nm. The FTIR analysis revealed important functional groups that were responsible for the reduction and stabilization of AgNPs. In the in vitro assays, 100 μg/ml of AgNPs and AgNPs-PE strongly inhibited Fusarium oxysporum. The same treatments tested against Fusarium sprayed on tomato plants in controlled environment exhibited nearly 100% plant survival with no observable phytotoxicity. These finding provide a simple baseline to control Fusarium wilt using silver nano bio-control agents without affecting the crop health.

Antagonist Effect of Bacillus Species against Alternaria Rot of Tomato

The PGPR possesses antifungal activity against a broad range of pathogens. In this research study, the LPs of Bacillus strain B1 and B2 against Alternaria alternata were assessed. In vitro and in Planta assay on tomato fruit was made to check the post-harvest quality of fruits. The lipopeptides produced by both strains suppress the growth of the fungal pathogen Alternaria alternata and lessen the disease severity of the Alternaria rot on tomato fruits. The result revealed the quality of the Bacillus-treated fruits was also maintained and satisfactory. The lipopeptides of bacillus strain B1 and B2 represent a new innovative method to lessen the use of fungicides and chemical approaches to reduce the post-harvest loss caused by Alternaria alternata.