Impact of synthetic chemicals and botanicals on bacterial leaf streak disease and wheat productivity parameters

Current research effort was directed focusing on evaluating the wheat germplasm for determination of source of resistance against the bacterial leaf streak (BLS) disease and the morpho-biochemical characterization of Xanthomonas translucens pv. undulosa (Xtu), the causative agent of bacterial leaf streak of wheat. The bacterium Xtu showed yellow, mucoid, circular and convex shaped growth when cultured on artificial growth media (NA). Gram staining, Catalase, KOH and Kovacs oxidase tests were employed that confirmed the Xtu as gram negative (-ve) bacterium. Fifteen wheat varieties/advanced lines were evaluated against BLS disease employing Randomized Complete Block Design (RCBD) to estimate the disease incidence over two years (2023 and 2024). Screening results for both years revealed that only one advanced line (PBG Line 8) expressed highly resistant response while two varieties PBG Line 1 and NARC-2008 exhibited resistant response. Two advanced lines (PBG Line 2 and PBG Line 5) were observed as moderately resistant while, moderately susceptible response was shown by FSD-2008, PBG Line 4, Ujala-2016, PBG Line 3, PBG Line 7 and PBG Line 6. The remaining varieties/advanced lines Umeed-2014, Zincol 2015, PBG Line 9 and PBG Line 10 showed susceptible response. The successful screening of wheat germplasm for resistance to bacterial leaf streak disease is an important source of resistance for breeding programs. Future research should prioritize leveraging this resource to develop enhanced cultivars and strengthen integrated management strategies for bacterial leaf streak disease (BLS). This approach will support the advancement of disease-resistant varieties and promote more effective, sustainable disease management solutions.

The genus Streptomyces demonstrates enormous promise in promoting plant growth and protecting plants against various pathogens. Single and consortium treatments of two selected Streptomyces strains (Streptomyces shenzhenensis TKSC3 and Streptomyces sp. SS8) were evaluated for their growth-promoting potential on rice, and biocontrol efficiency through induced systemic resistance (ISR) mediation against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS) disease. Seed bacterization by Streptomyces strains improved seed germination and vigor, relative to the untreated seed. Under greenhouse conditions, seed bacterization with consortium treatment TKSC3 + SS8 increased seed germination, root length, and dry weight by 20%, 23%, and 33%, respectively. Single and consortium Streptomyces treatments also successfully suppressed Xoc infection. The result was consistent with defense-related enzyme quantification wherein single and consortium Streptomyces treatments increased peroxidase (POX), polyphenol oxidase, phenylalanine ammonia-lyase, and β,1–3 glucanase (GLU) accumulation compared to untreated plant. Within all Streptomyces treatments, consortium treatment TKSC3 + SS8 showed the highest disease suppression efficiency (81.02%) and the lowest area under the disease progress curve value (95.79), making it the best to control BLS disease. Consortium treatment TKSC3 + SS8 induced the highest POX and GLU enzyme activities at 114.32 μmol/min/mg protein and 260.32 abs/min/mg protein, respectively, with both enzymes responsible for plant cell wall reinforcement and resistant interaction. Our results revealed that in addition to promoting plant growth, these Streptomyces strains also mediated ISR in rice plants, thereby, ensuring protection from BLS disease.

Studies were conducted to characterize spatial and temporal progress of bacterial leaf streak disease (Xanthomonas translucens pv. translucens) on susceptible (Florida 304) and moderately resistant (Terral 101) winter wheat cultivars. Epidemics were initiated with rifampicin-resistant strain 88-14rif of X. translucens pv. translucens by establishing point sources of inoculum in plot centers. Incidence of bacterial leaf streak was assessed five times in 1995 and three times in 1996, starting from the first observation of leaf streak symptoms. Rainfall, temperature, and wind speed were significantly related to disease incidence, but relative humidity was not. The Gompertz model gave the best statistical fit for the progression of disease incidence over time. Average rates of disease progress (k) obtained from the regression of bacterial leaf streak incidence against time provided a good method of comparing the cultivars Florida 304 and Terral 101 and were consistent across locations. Bacterial leaf streak disease gradients were best described by the negative exponential model. Bacterial leaf streak incidence decreased with distance from inoculum source for both cultivars. Disease incidence on Terral 101 was near 0% at 2 m from the source, and disease incidence close to the source was consistently lower on Terral 101 than on Florida 304 at all growth stages sampled. This was not unexpected because the two cultivars differed in susceptibility. Disease incidence data were more useful than severity data in providing a good estimate of disease spread away from the source.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 μL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

Rice is one of the most important food crops in the world and is of vital importance to many countries. Various diseases caused by fungi, bacteria and viruses constantly threaten rice plants and cause yield losses. Bacterial leaf streak disease (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most devastating rice diseases. However, most modern rice varieties are susceptible to BLS. In this study, we applied the QTL-seq approach using an F2 population derived from the cross between IR62266 and Homcholasit (HSC) to rapidly identify the quantitative trait loci (QTL) that confers resistance to BLS caused by a Thai Xoc isolate, SP7-5. The results showed that a single genomic region at the beginning of chromosome 5 was highly associated with resistance to BLS. The gene xa5 was considered a potential candidate gene in this region since most associated single nucleotide polymorphisms (SNPs) were within this gene. A Kompetitive Allele-Specific PCR (KASP) marker was developed based on two consecutive functional SNPs in xa5 and validated in six F2 populations inoculated with another Thai Xoc isolate, 2NY2-2. The phenotypic variance explained by this marker (PVE) ranged from 59.04% to 70.84% in the six populations. These findings indicate that xa5 is a viable candidate gene for BLS resistance and may help in breeding programs for BLS resistance.

In 2014, researchers in Uganda spotted signs typical bacterial leaf streak disease (Xanthomonas oryzae pv. oryzicola) in rice fields in Eastern Uganda. The disease was later confirmed to be bacterial leaf streak. In order to effectively plan for measures to manage this potentially devastating disease, it was imperative to score rice germplasm in Uganda for reaction to Xoc. Eighty four genotypes from the National Rice Improvement program were evaluated for their reaction to BLS using two Xoc isolates collected from Namulonge and Iganga. These were inoculated by the infiltration method using a needless syringe 30 days after planting. Data were collected on the streak length induced by BLS on the leaves 15 days after inoculation. The mean streak length per genotype was interpreted as; Resistant (R), 030mm. Genotypes showed signficant variability (P Xoc isolates reacted signficantly differently (P=0.011) on the rice genotypes. For the Iganga isolate, 6 genotypes were resistant while 17 were moderately resistant. For the Namulonge isolate, 3 genotypes were resistant while 7 were moderately resistant. Three genotypes were resistant to both isolates. The observations ranged from highly resistant in Nerica1 to highly susceptible in Du 363. The resistant genotypes identified could be used as sources of genes for introgression into susceptible but agronomically desirable genotypes.

Xanthomonas translucens pv. undulosa (Xtu) is a proteobacteria which causes bacterial leaf streak (BLS) or bacterial chaff disease in wheat and barley. The constant competition for zinc (Zn) metal nutrients contributes significantly in plant–pathogen interactions. In this study, we have employed a systematic in silico approach to study the Zn-binding proteins of Xtu. From the whole proteome of Xtu, we have identified approximately 7.9% of proteins having Zn-binding sequence and structural motifs. Further, 115 proteins were found homologous to plant–pathogen interaction database. Among these 115 proteins, 11 were predicted as putative secretory proteins. The functional diversity in Zn-binding proteins was revealed by functional domain, gene ontology and subcellular localization analysis. The roles of Zn-binding proteins were found to be varied in the range from metabolism, proteolysis, protein biosynthesis, transport, cell signalling, protein folding, transcription regulation, DNA repair, response to oxidative stress, RNA processing, antimicrobial resistance, DNA replication and DNA integration. This study provides preliminary information on putative Zn-binding proteins of Xtu which may further help in designing new metal-based antimicrobial agents for controlling BLS and bacterial chaff infections on staple crops.

Efficacy of organic disease control products on common foliar diseases of tomato in field and greenhouse trials

Bacterial leaf streak (BLS) is one of the internationally significant quarantine diseases in rice. Effectively utilizing BLS resistance genes from wild rice (Oryza rufipogon Griff.) to breed new varieties offers a fundamental solution for BLS control. This study focused on the fine mapping of the BLS resistance gene bls2 and the development of closely linked molecular markers for breeding BLS-resistant lines. Using a Guangxi common wild rice accession DY19 (carrying bls2) as the donor parent and the highly BLS-susceptible indica rice variety 9311 as the recipient parent, BLS-resistant rice lines were developed through multiple generations of backcrossing and selfing, incorporating molecular marker-assisted selection (MAS), single nucleotide polymorphism(SNP) chip genotyping, pathogen inoculation assays, and agronomic trait evaluation. The results showed that bls2 was delimited to a 113 kb interval between the molecular markers ID2 and ID5 on chromosome 2, with both markers exhibiting over 98% accuracy in detecting bls2. Four stable new lines carrying the bls2 segment were obtained in the BC5F4 generation. These four lines showed highly significant differences in BLS resistance compared with 9311, demonstrating moderate resistance or higher with average lesion lengths ranging from 0.69 to 1.26 cm. Importantly, no significant differences were observed between these resistant lines and 9311 in key agronomic traits, including plant height, number of effective panicles, panicle length, seed setting rate, grain length, grain width, length-to-width ratio, and 1000-grain weight. Collectively, two molecular markers closely linked to bls2 were developed, which can be effectively applied in MAS, and four new lines with significantly enhanced resistance to BLS and excellent agronomic traits were obtained. These findings provide technical support and core germplasm resources for BLS resistance breeding.

This study provides a phylogeographic insight into the population diversity of Xanthomonas translucens strains causing bacterial leaf streak disease of small-grain cereals in Iran. Among the 65 bacterial strains isolated from wheat, barley, and gramineous weeds in eight Iranian provinces, multilocus sequence analysis and typing (MLSA and MLST) of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD), identified 57 strains as X. translucens pv. undulosa, while eight strains were identified as X. translucens pv. translucens. Although the pathogenicity patterns on oat and ryegrass weed species varied among the strains, all X. translucens pv. undulosa strains were pathogenic on barley, Harding's grass, rye (except for XtKm35) and wheat, and all X. translucens pv. translucens strains were pathogenic on barley and Harding's grass, while none of the latter group was pathogenic on rye or wheat (except for XtKm18). MLST using the 65 strains isolated in Iran, as well as the sequences of the four genes from 112 strains of worldwide origin retrieved from the GenBank database, revealed higher genetic diversity (i.e., haplotype frequency, haplotype diversity, and percentage of polymorphic sites) among the Iranian population of X. translucens than among the North American strains of the pathogen. High genetic diversity of the BLS pathogen in Iran was in congruence with the fact that the Iranian Plateau is considered the center of origin of cultivated wheat. However, further studies using larger collections of strains are warranted to precisely elucidate the global population diversity and center of origin of the pathogen.IMPORTANCE Bacterial leaf streak (BLS) of small-grain cereals (i.e., wheat and barley) is one of the economically important diseases of gramineous crops worldwide. The disease occurs in many countries across the globe, with particular importance in regions characterized by high levels of precipitation. Two genetically distinct xanthomonads-namely, Xanthomonas translucens pv. undulosa and X. translucens pv. translucens-have been reported to cause BLS disease on small-grain cereals. As seed-borne pathogens, the causal agents are included in the A2 list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Despite its global distribution and high economic importance, the population structure, genetic diversity, and phylogeography of X. translucens remain undetermined. This study, using MLSA and MLST, provides a global-scale phylogeography of X. translucens strains infecting small-grain cereals. Based on the diversity parameters, neutrality indices, and population structure, we observe higher genetic diversity of the BLS pathogen in Iran, which is geographically close to the center of origin of common wheat, than has so far been observed in other areas of the world, including North America. The results obtained in this study provide a novel insight into the genetic diversity and population structure of the BLS pathogen of small-grain cereals on a global scale.

Evaluation of wheat genotypes for resistance to bacterial leaf streak caused by Xanthomonas translucens pv. undulosa in field and greenhouse trials

Xanthomonas translucens, the causal agent of bacterial leaf streak disease (BLS) in cereals, is a re-emerging pathogen that is becoming increasingly destructive across the world. While BLS has caused yield losses in the past, there is anecdotal evidence that newer isolates may be more virulent. We observed that two X. translucens isolates collected from two sites in Colorado, USA, are more aggressive on current wheat and barley varieties compared to older isolates, and we hypothesize that genetic changes between recent and older isolates contribute to the differences in isolate aggressiveness. To test this, we phenotyped and genetically characterized two X. translucens isolates collected from Colorado in 2018, which we designated CO236 (from barley) and CO237 (from wheat). Using pathovar-specific phenotyping and PCR primers, we determined that CO236 belongs to pathovar translucens (Xtt) and CO237 belongs to pathovar undulosa (Xtu). We sequenced the full genomes of the isolates using Oxford Nanopore long-read sequencing, and compared their whole genomes against published X. translucens genomes. This analysis confirmed our pathovar designations for Xtt CO236 and Xtu CO237, and showed that, at the whole-genome level, there were no obvious genomic structural changes between Xtt CO236 and Xtu CO237 and other respective published pathovar genomes. Focusing on pathovar undulosa (Xtu CO237), we then compared putative type III effectors among all available Xtu isolate genomes and found that they were highly conserved. However, there were striking differences in the presence and sequence of various transcription activator-like effectors between Xtu CO237 and published undulosa genomes, which correlate with isolate virulence. Here, we explore the potential implications of the differences in these virulence factors, and provide possible explanations for the increased virulence of recently emerged isolates.

Five chemicals, incuding Flare, Plant Protector, Mancozeb, Agrimycine, and Copper oxychloride, and five plant extracts including N. tabacum, A. indica, M. oleifera, D. alba and C. longa were evaluated against bacterial blight of cotton caused by Xanthomonas citri pv. malvacearum (a bacterium). The impact of chemicals and plant extracts on bacterial development was tested in laboratory while on disease reduction was tested in green house and field experiments. Laboratory experiments showed that maximum inhibition zone of bacterial growth was expressed by Flare (1.693cm) at all concentrations followed by Plant Protector (1.473 cm), Mancozeb (1.290 cm), Agrimycine (1.150 cm) and copper oxy-chloride (0.953) cm respectively while in case of plant extracts maximum inhibition was expressed by N. tabacum (0.650 cm) followed by A. indica (0.486), M. oleifera (0.350), D. alba (0.256 cm) and C. longa (0.168 cm). Green house experiment revealed that the best result was produced by the combination of Flare and N. tabacum by indicating lowest disease incidence (32.27%) at all the tested concentration. Same results were obtained in field experiment, where the lowest disease incidence (40.41%) was recorded when the ,Flare and N. tabacum were applied in combination although it was higher then green house. This study concludes that N. tabacum and Flare are better option against bacterial disease development and even their combination is more significant lowering the bacterial blight disease incidence on cotton. Selection of suitable formulation and method of application could be the future aspects of plant product especially N. tabacum related research.

In 2011, the bacterial leaf streak disease of the monocotyledonous flowering plant, commonly known as bird of paradise (Strelitzia reginae), occurred in a nursery in Guangzhou, Guangdong Province, China. Lesions on diseased leaves began as water-soaked leaf spots or streaks near the central and secondary veins, eventually expanded along veins and became brown necrotic streaks. Occasionally, during wet conditions, seedlings were completely blighted. The disease incidence was about 12% in the nursery. Bacteria were consistently isolated on nutrient agar (NA) (4) from surface-sterilized symptomatic lesions and purified on NA. Three bacterial strains were tested for pathogenicity on S. reginae plants. Three plants were inoculated per bacterial strain (bacterial suspensions 107 CFU/ml in nutrient broth [NB] [4]) by wounding three young, fully expanded leaves (four wounds per leaf) with needle. Plants were placed in polyethylene bags 1 day before inoculation and maintained for 7 days after inoculation. Three control plants were inoculated with NB. Water-soaked areas on leaves were observed on all inoculated plants 7 days after inoculation. Within 10 days, brown streaks were observed. All strains induced similar symptoms as those observed on the plants in the nursery. Control plants showed no symptoms. For molecular identification, a near full-length sequence of the 16S rRNA gene was amplified from strain TNT1-1 (GenBank Accession No. JX901049.1) with primers 27F and 1492R (3), obtaining a PCR product of ~1,500 bp. A BLAST search in GenBank revealed the highest similarity (99.5%) to sequences of Burkholderia cepacia (FN178432.1 and FN178432.1). BIOLOG identification showed that TTN1-1 had the highest probability index of 0.85 and highest similarity index of 0.85 to B. cepacia. For biochemical characteristics, the strain was gram negative, anaerobic growth test negative, oxidase negative, catalase positive, did not produce fluorescent pigment on KB (4), did not grow on DIM agar (4), arginine dihydrolysis negative, nitrate reduction negative, starch hydrolysis negative, gelatin liquefaction negative, citrate, D-arabinose, L-fructose, trehalose, and maltose utilization positive, didn't produce acid from glucose, and grew on Tween 80 medium at 41°C. The above characteristics were identical to that of reference isolate B. cepacia ATCC 25416. Additionally, bacteria isolated on NA from the leading edge of lesions of inoculated plants with the strain were identical to the inoculated strain based on 16S rDNA sequence analysis, but no bacteria were recovered from the wounded sites on the control plants. Therefore, bacterial leaf streak of bird of paradise is caused by B. cepacia based on Koch's postulates. In contrast, two bacterial diseases on S. reginae were previously reported to be caused by Xanthomonas campestris (1) and B. gladioli (2) in the United States and Italy, respectively. A similar leaf streak disease on S. nicolai was caused by Acidovorax avenae subsp. avenae in the United States (5). To our knowledge, this is the first report of a leaf streak disease on S. reginae caused by B. cepacia.

Wheat (Triticum aestivum) loses 21.5% yield to pests and diseases annually (Savary et al. 2019). Among the wheat diseases, bacterial leaf streak (BLS) is a growing problem, costing $78.5 million in losses (https://cropprotectionnetwork.org/). In July 2022, we sampled winter wheat leaf samples at Volga (44.30, -96.92), South Dakota, USA with an estimated disease incidence of 40% (n=100). The typical symptoms were water-soaking with large necrotic and chlorotic streaks extending the length of the leaves and were strikingly similar to BLS. To isolate the pathogen, leaves were cut lengthwise into 1 cm pieces and surface-sterilized using a 10% NaOCl solution for 3 min, followed by 70% ethanol for 3 min, and then rinsed with sterile distilled water and placed in 500 ul of sterile distilled water for 5 min and using a sterile loop the water was streaked over a plate of Nutrient Agar (NA). Following Duveiller et al. (1997), the streaked plate was incubated in the dark at 28℃ for 48 h. Observed single colonies were sub-cultured thrice onto fresh NA plates to obtain a pure culture. We named the culture SD101. Bacteria were found to be gram-negative with a colony morphology initially raised, smooth, and white that later turned yellow. DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's protocol, and sequenced using Nanopore MinION R9.4 (Oxford Nanopore Technology). We used the Rapid Annotation Using Subsystems Technology approach (Aziz eal. 2008) to extract the 16S rDNA, DNA gyrase subunit B (gyrB), and translation initiation factor IF-2 (infB) gene sequences that were deposited in GenBank under accession numbers PP329908.1 for 16S rDNA, PP496481 for infB, and PP328920.1 for gyrB. Homology analysis using CLC Genomics Workbench 22.0.2 (QIAGEN) and BLASTn against the GenBank nucleotide database resulted in a 99.74% match (1543/ 1547 bp) of the 16S sequence, 99.59% match (2674/ 2685 bp) of the infB sequence, and 99.42% match (2396/ 2410 bp) of the gyrB sequence with Pantoea ananatis strain AJ13355 (AP012032). To test pathogenicity, seeds of spring wheat breeding line SD4892 were planted in 30 cm × 30 cm pots in a greenhouse under a 16 h light photoperiod. The inoculum was prepared from 48-h-old NA plates of SD101 rinsed with 1X Phosphate Buffer Saline (PBS buffer), adjusted to an OD600 = 1.0, and amended with two drops of Tween 20 (polyoxyethylene sorbitol ester, Millipore Sigma). PBS with Tween 20 was used as a negative control. The inoculum was sprayed on 15 replicates of 15-day-old seedlings, kept at 95% relative humidity for 48 h, then moved to the greenhouse at 23 to 25°C. The symptoms appeared as water soaking that later turned to necrotic streaks with surrounding chlorosis on all 15 inoculated plants while control plants remained healthy. The pathogen was re-isolated from the leaves as described above. The 16S rDNA, infB, and gyrB sequences amplified and sequenced were identical to the gene sequences from the whole genome sequencing. The experiment was repeated with the same results, completing Koch's postulates. Although P. ananatis is pathogenic on corn, rice, and other plant species in the United States (Coutinho et al. 2009), and was reported pathogenic on wheat in Poland (Krawczyk et al. 2020), this is the first report of its pathogenicity on wheat in the United States. The prevalence, and incidence of BLS disease caused by P. ananatis in wheat is needed to estimate its threat to wheat production and to develop management strategies.