Deciphering fungicide resistance in Phytophthora: mechanisms, prevalence, and sustainable management approaches.

The genus Phytophthora contains more than 100 plant pathogenic species that parasitize a wide range of plants, including economically important fruits, vegetables, cereals, and forest trees, causing significant losses. Global agriculture is seriously threatened by fungicide resistance in Phytophthora species, which makes it imperative to fully comprehend the mechanisms, frequency, and non-chemical management techniques related to resistance mutations. The mechanisms behind fungicide resistance, such as target-site mutations, efflux pump overexpression, overexpression of target genes and metabolic detoxification routes for fungicides routinely used against Phytophthora species, are thoroughly examined in this review. Additionally, it assesses the frequency of resistance mutations in various Phytophthora species and geographical areas, emphasizing the rise of strains that are resistant to multiple drugs. The effectiveness of non-chemical management techniques, including biological control, host resistance, integrated pest management plans, and cultural practices, in reducing fungicide resistance is also thoroughly evaluated. The study provides important insights for future research and the development of sustainable disease management strategies to counter fungicide resistance in Phytophthora species by synthesizing current information and identifying knowledge gaps.

Unveiling the Genetic Tapestry: Exploring Rhizoctonia solani AG-3 Anastomosis Groups in Potato Crops across Borders.

The current study was carried out to screen 10 isolates (ARS-01-ARS-10) of Rhizoctonia. solani from potato tubers cv. Kuroda, which were collected from various potato fields in Multan, Pakistan. The isolates were found to be morphologically identical, as the hyphae exhibit the production of branches at right angles and acute angles often accompanied by septum near the emerging branches. Anastomosis grouping showed that these isolates belonged to AG-3. A pathogenicity test was performed against the susceptible Kuroda variety and among the isolates, ARS-05 exhibited the highest mean severity score of approximately 5.43, followed by ARS-09, which showed a mean severity score of about 3.67, indicating a moderate level of severity. On the lower end of the severity scale, isolates ARS-06 and ARS-07 displayed mean severity scores of approximately 0.53 and 0.57, respectively, suggesting minimal symptom severity. These mean severity scores offer insights into the varying degrees of symptom expression among the different isolates of R. solani under examination. PCoA indicates that the severe isolate causing black scurf on the Kuroda variety was AG-3. A comprehensive analysis of the distribution, genetic variability, and phylogenetic relationships of R. solani anastomosis groups (AGs) related to potato crops across diverse geographic regions was also performed to examine AG prevalence in various countries. AG-3 was identified as the most widespread group, prevalent in Sweden, China, and the USA. AG-5 showed prominence in Sweden and the USA, while AG-2-1 exhibited prevalence in China and Japan. The phylogenetic analysis unveiled two different clades: Clade I comprising AG-3 and Clade II encompassing AG-2, AG-4, and AG-5, further subdivided into three subclades. Although AGs clustered together regardless of origin, their genetic diversity revealed complex evolutionary patterns. The findings pave the way for region-specific disease management strategies to combat R. solani's impact on potato crops.

Molecular detection of Babesia ovis and blood parameters' investigation reveal hematological and biochemical alterations in babesiosis-infected Lohi sheep in Multan, Pakistan.

Background: Babesia infections in sheep can cause a wide range of clinical and laboratory presentations. Changes in blood parameters are a meaningful manifestation of physiological and pathological changes in an organism. Aim: Therefore, the present study was conducted to analyze and compare hematological and biochemical parameters between blood profiles of Lohi sheep naturally infected and uninfected with Babesia ovis, the main causative agent of ovine babesiosis. Methods: Initially, blood and serum samples from 67 Lohi sheep were collected, DNA was extracted and babesial infection was detected through polymerase chain reaction. The overall infection rate of B. ovis was 37% (25/67). Sixteen infected (experiment group) and 16 uninfected (control group) sheep that were apparently healthy with no history of previous treatment for babesiosis, were selected for hemato-biochemical analysis. Blood samples were analyzed through an automatic CBC analyzer, while serum collected from gel vacutainers was analyzed for blood urea, blood urea nitrogen (BUN), creatinine, and total bilirubin. Each parameter was compared between infected and uninfected animals using a paired t-test in Minitab Express™ software for statistical analyses. Results: Erythron comparison showed a highly significant ( p < 0.0001) decrease in RBC, hemoglobin, and Hct. A nonsignificant increase in mean corpuscular volume (MCV), red cell distribution width (RDW), and RDW-standard deviation (RDW-SD), while a nonsignificant decrease in mean corpuscular haemoglobin (MCH) and MCH concentration (MCHC) values was recorded in infected sheep. Leukon comparison showed a significantly low level of total leukocyte (p < 0.001) in infected sheep. Platelet (Plt) along with platelet crit (Pct) and platelet distribution width (PDW) were nonsignificantly higher, whereas a nonsignificant decrease in mean Plt volume was recorded in infected sheep as compared to uninfected animals. Among biochemical parameters, blood urea, BUN, and total bilirubin showed significant differences (p < 0.05), while creatinine showed a nonsignificant difference. Conclusion: To the best of our knowledge, this is the first report on hemato-biochemical changes associated with babesiosis in the Lohi breed. Consistent with hemolytic anemia, these data would justify physical examination and, together with the medical history, would provide an excellent basis for the diagnosis of babesiosis.

Unlocking the secrets of soil microbes: How decades-long contamination and heavy metals accumulation from sewage water and industrial effluents shape soil biological health.

Heavy metals contamination is posing severe threat to the soil health and environmental sustainability. Application of industrial and sewage waste as irrigation and growing urbanization and agricultural industry is the main reason for heavy metals pollution. Therefore, the present study was planned to assess the influence of different irrigation sources such as industrial effluents, sewage wastewater, tube well water, and canal water on the soil physio-chemical, soil biological, and enzymatic characteristics. Results showed that sewage waste and industrial effluents affect the soil pH, organic matter, total organic carbon, and cation exchange capacity. The highest total nickel (383.71 mg kg-1), lead (312.46 mg kg-1), cadmium (147.75 mg kg-1), and chromium (163.64 mg kg-1) were recorded with industrial effluents application. Whereas, industrial effluent greatly reduced the soil microbial biomass carbon (SMB-C), soil microbial biomass nitrogen (SMB-N), soil microbial biomass phosphorus (SMB-P), and soil microbial biomass sulphur (SMB-S) in the winter season at sowing time. Industrial effluent and sewage waste inhibited the soil enzymes activities. For instance, the minimum activity of amidase, urease, alkaline-phosphatase, ß-glucosidase, arylsulphatase and dehydrogenase activity was noted with HMs contamination. The higher levels of metals accumulation was observed in vegetables grown in soil contaminated with untreated waste water and industrial effluent in comparison to soil irrigated with canal and tube well water. The mean increase in soil microbial parameters and enzyme activities was also observed in response to the change in season from winter to spring due to increase in soil mean temperature. The SMB-C, SMB-N, SMB-P and SMB-S showed significant positive correlation with soil enzymes (amidase, urease, alkaline-phosphatase, ß-glucosidase, arylsulphatase and dehydrogenase). The heavy metals accumulation in soil is toxic to microorganisms and inhibits enzyme functions critical for nutrient cycling and organic matter decomposition and can disrupt the delicate balance of soil ecosystem and may lead to long-term damage of soil biological health.

Assessment of Genetic Variability and Evolutionary Relationships of Rhizoctonia solani Inherent in Legume Crops.

Rhizoctonia solani is one of the most common soil-borne fungal pathogens of legume crops worldwide. We collected rDNA-ITS sequences from NCBI GenBank, and the aim of this study was to examine the genetic diversity and phylogenetic relationships of various R. solani anastomosis groups (AGs) that are commonly associated with grain legumes (such as soybean, common bean, pea, peanut, cowpea, and chickpea) and forage legumes (including alfalfa and clover). Soybean is recognized as a host for multiple AGs, with AG-1 and AG-2 being extensively investigated. This is evidenced by the higher representation of sequences associated with these AGs in the NCBI GenBank. Other AGs documented in soybean include AG-4, AG-7, AG-11, AG-5, AG-6, and AG-9. Moreover, AG-4 has been extensively studied concerning its occurrence in chickpea, pea, peanut, and alfalfa. Research on the common bean has been primarily focused on AG-2, AG-4, and AG-1. Similarly, AG-1 has been the subject of extensive investigation in clover and cowpea. Collectively, AG-1, AG-2, and AG-4 have consistently been identified and studied across these diverse legume crops. The phylogenetic analysis of R. solani isolates across different legumes indicates that the distinct clades or subclades formed by the isolates correspond to their specific anastomosis groups (AGs) and subgroups, rather than being determined by their host legume crop. Additionally, there is a high degree of sequence similarity among isolates within the same clade or subclade. Principal coordinate analysis (PCoA) further supports this finding, as isolates belonging to the same AGs and/or subgroups cluster together, irrespective of their host legume. Therefore, the observed clustering of R. solani AGs and subgroups without a direct association with the host legume crop provides additional support for the concept of AGs in understanding the genetic relationships and evolution of R. solani.

Corrigendum: Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches.

[This corrects the article DOI: 10.3389/fpls.2022.1009395.].

Nanopesticides in comparison with agrochemicals: Outlook and future prospects for sustainable agriculture.

Agrochemicals are products of advanced technologies that use inorganic pesticides and fertilizers. Widespread use of these compounds has adverse environmental effects, leading to acute and chronic exposure. Globally, scientists are adopting numerous green technologies to ensure a healthy and safe food supply and a livelihood for everyone. Nanotechnologies significantly impact all aspects of human activity, including agriculture, even if synthesizing certain nanomaterials is not environmentally friendly. Numerous nanomaterials may therefore make it easier to create natural insecticides, which are more effective and environmentally friendly. Nanoformulations can improve efficacy, reduce effective doses, and extend shelf life, while controlled-release products can improve the delivery of pesticides. Nanotechnology platforms enhance the bioavailability of conventional pesticides by changing kinetics, mechanisms, and pathways. This allows them to bypass biological and other undesirable resistance mechanisms, increasing their efficacy. The development of nanomaterials is expected to lead to a new generation of pesticides that are more effective and safer for life, humans, and the environment. This article aims to express at how nanopesticides are being used in crop protection now and in the future. This review aims to shed some light on the various impacts of agrochemicals, their benefits, and the function of nanopesticide formulations in agriculture.

Enhanced Production of Active Photosynthetic and Biochemical Molecules in Silybum marianum L. Using Biotic and Abiotic Elicitors in Hydroponic Culture.

Elicitors are stressors that activate secondary pathways that lead to the increased production of bioactive molecules in plants. Different elicitors including the fungus Aspergillus niger (0.2 g/L), methyl jasmonate (MeJA, 100 µM/L), and silver nanoparticles (1 µg/L) were added, individually and in combination, in a hydroponic medium. The application of these elicitors in hydroponic culture significantly increased the concentration of photosynthetic pigments and total phenolic contents. The treatment with MeJA (methyl jasmonate) (100 µM/L) and the co-treatment of MeJA and AgNPs (silver nanoparticles) (100 µM/L + 1 µg/L) exhibited the highest chlorophyll a (29 µg g-1 FW) and chlorophyll b (33.6 µg g-1 FW) contents, respectively. The elicitor MeJA (100 µM/L) gave a substantial rise in chlorophyll a and b and total chlorophyll contents. Likewise, a significant rise in carotenoid contents (9 µg/g FW) was also observed when subjected to meJA (100 µM/L). For the phenolic content, the treatment with meJA (100 µM/L) proved to be very effective. Nevertheless, the highest production (431 µg/g FW) was observed when treated with AgNPs (1 µg/L). The treatments with various elicitors in this study had a significant effect on flavonoid and lignin content. The highest concentration of flavonoids and lignin was observed when MeJA (100 mM) was used as an elicitor, following a 72-h treatment period. Hence, for different plant metabolites, the treatment with meJA (100 µM/L) and a co-treatment of MeJA and AgNPs (100 µM/L + 1 µg/L) under prolonged exposure times of 120-144 h proved to be the most promising in the accretion of valuable bioactive molecules. The study opens new insights into the use of these elicitors, individually or in combination, by using different concentrations and compositions.

Population Genetics and Anastomosis Group's Geographical Distribution of Rhizoctonia solani Associated with Soybean.

Rhizoctonia solani is a species complex composed of many genetically diverse anastomosis groups (AG) and their subgroups. It causes economically important diseases of soybean worldwide. However, the global genetic diversity and distribution of R. solani AG associated with soybean are unknown to date. In this study, the global genetic diversity and distribution of AG associated with soybean were investigated based on rDNA-ITS sequences deposited in GenBank and published literature. The most prevalent AG, was AG-1 (40%), followed by AG-2 (19.13%), AG-4 (11.30%), AG-7 (10.43%), AG-11 (8.70%), AG-3 (5.22%) and AG-5 (3.48%). Most of the AG were reported from the USA and Brazil. Sequence analysis of internal transcribed spacers of ribosomal DNA separated AG associated with soybean into two distinct clades. Clade I corresponded to distinct subclades containing AG-2, AG-3, AG-5, AG-7 and AG-11. Clade II corresponded to subclades of AG-1 subgroups. Furthermore, AG and/or AG subgroups were in close proximity without corresponding to their geographical origin. Moreover, AG or AG subgroups within clade or subclades shared higher percentages of sequence similarities. The principal coordinate analysis also supported the phylogenetic and genetic diversity analyses. In conclusion, AG-1, AG-2, and AG-4 were the most prevalent AG in soybean. The clade or subclades corresponded to AG or AG subgroups and did not correspond to the AG's geographical origin. The information on global genetic diversity and distribution will be helpful if novel management measures are to be developed against soybean diseases caused by R. solani.

Green Chemistry Based Synthesis of Zinc Oxide Nanoparticles Using Plant Derivatives of Calotropis gigantea (Giant Milkweed) and Its Biological Applications against Various Bacterial and Fungal Pathogens.

Nanotechnology is a burning field of scientific interest for researchers in current era. Diverse plant materials are considered as potential tool in green chemistry based technologies for the synthesis of metal nanoparticles (NPs) to cope with the hazardous effects of synthetic chemicals, leading to severe abiotic climate change issues in today's agriculture. This study aimed to determine the synthesis and characterization of metal-based nanoparticles using extracts of the selected plant Calotropis gigantea and to evaluate the enzyme-inhibition activities and antibacterial and antifungal activity of extracts of metal-based zinc nanoparticles using C. gigantea extracts. The crystal structure and surface morphology were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). C. gigantea was examined for antimicrobial activity against clinical isolates of bacteria and fungi. The water, ethanolic, and acetone extracts of C. gigantea were studied for their antagonistic action against bacterial strains (E. coli, S. aureus, P. multocida, and B. subtilis) and selected fungal strains (A. paracistic, F. solani, A. niger, S. ferrugenium, and R. nigricans). In vitro antimicrobial activity was determined by the disc diffusion method, where C. gigantea wastested for AChE and BChE inhibitory activity using Ellman's methodology. The kinetic analysis was performed by the proverbial Berthelot reaction for urease inhibition. The results showed that out of all the extracts tested, ethanolic and water extracts possessed zinc nanoparticles. These extracts showed the maximum zone of inhibition against F. solani and P. multocida and the lowest against S. ferrugenium and B. subtilis. A potential source of AChE inhibitors is certainly provided by the abundance of plants in nature. Numerous phyto-constituents, such as AChE and BChE inhibitors, have been reported in this communication. Water extract was active and has the potential for in vitro AChE and BChE inhibitory activity. The urease inhibition with flower extracts of C. gigantea revealed zinc nanoparticles in water extracts that competitively inhibited urease enzymes. In the case of cholinesterase enzymes, it was inferred that the water extract and zinc nanoparticles have more potential for inhibition of BChE than AChE and urease inhibition. Furthermore, zinc nanoparticles with water extract are active inthe inhibition of the bacterial strains E. coli, S. aureus, and P. multocida and the fungal strains A. paracistic, F. solani, and A. niger.

Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches.

Plant tissue culture technique employed for the identification and isolation of bioactive phytocompounds has numerous industrial applications. It provides potential benefits for different industries which include food, pharmaceutical and cosmetics. Various agronomic crops i.e., cereals, fruits, vegetables, ornamental plants and forest trees are currently being used for in vitro propagation. Plant tissue culture coupled with biotechnological approaches leads towards sustainable agricultural development providing solutions to major food security issues. Plants are the rich source of phytochemicals with medicinal properties rendering them useful for the industrial production of pharmaceuticals and nutraceuticals. Furthermore, there are numerous plant compounds with application in the cosmetics industry. In addition to having moisturizing, anti-ageing, anti-wrinkle effects; plant-derived compounds also possess pharmacological properties such as antiviral, antimicrobial, antifungal, anticancer, antioxidant, anti-inflammatory, and anti-allergy characteristics. The in vitro propagation of industrially significant flora is gaining attention because of its several advantages over conventional plant propagation methods. One of the major advantages of this technique is the quick availability of food throughout the year, irrespective of the growing season, thus opening new opportunities to the producers and farmers. The sterile or endangered flora can also be conserved by plant micro propagation methods. Hence, plant tissue culture is an extremely efficient and cost-effective technique for biosynthetic studies and bio-production, biotransformation, or bioconversion of plant-derived compounds. However, there are certain limitations of in-vitro plant regeneration system including difficulties with continuous operation, product removal, and aseptic conditions. For sustainable industrial applications of in-vitro regenerated plants on a large scale, these constraints need to be addressed in future studies.

First report of Fusarium equiseti causing leaf spots of Bitter gourd (Momordica charantia) in Pakistan.

Bitter gourd (Momordica charantia L.) is an important vegetable crop of the Cucurbitaceae family widely cultivated in Pakistan and around the world. In October 2020, a nutrition management trial of Bitter gourd cv. Seminis-200) was conducted on an area of 10,860 sq. ft. (99×110 feet) at the Agricultural Research farm of Bahauddin Zakariya University, Multan (30.2601° N, 71.5158° E), Pakistan. Symptoms of large, brown necrotic leaf spots were observed on the leaves of bitter gourd vines. The disease started from the yellowing of leaves within the reticulate venation and turned brown. Irregular brown leaf spots coalesced to form large necrotic areas followed by foliar chlorosis then wilting that occurred very late. There were no crown rot symptoms although there was slight discoloration of roots and when cut longitudinally, browning of tissues was observed. The disease was assessed visually with 37% incidence which resulted in poor quality and yield in terms of reduced size and yellowing of fruit. Infected vines along with the roots were collected for the isolation of pathogen. A total of 34 leaves and 22 root samples were collected from the field for isolation. The leaf, collar and root portions were cut into 0.5 to 1 cm in length and surface disinfected with 1% sodium hypochlorite (NaOCl) for 2-3 minutes followed by washing twice with autoclaved distilled water and after drying, placed on potato dextrose agar (PDA) medium, and incubated at 25±2 °C for one week. The fungal colonies of fluffy white growth with light orange pigment were isolated. For morphological characterization, a total of 4 pure cultures were isolated from leaves, collar region and root by single spore technique on carnation leaf agar (CLA) medium after 15 days of incubation at 25±2℃. Curved and thick-walled macroconidia with elongated or pointed apical characteristic foot-shaped basal cells were produced in sporodochia. Macroconidia with 5-7 septa measured 22.50-41.80 µm × 2.90-4.20 µm (n = 60). Thick, brown with roughened walls and subglobose ellipsoidal chlamydospores were observed in clumps or chains with the dimension of 5.8 to 10.8 µm (n = 20). On morphological characteristics, the fungus was identified as Fusarium equiseti (Corda) Sacc. according to Leslie and Summerell (2006). Two single spore isolates were used for molecular identification by amplifying ribosomal DNA of the internal transcribed spacer (ITS) region with ITS1/ITS4 primers (White et al. 1990) and for ß-tubulin gene region, primers T1/Bt-2b (O'Donnell and Cigelnik, 1997) were used. The obtained sequences were deposited in GenBank with accession numbers MW880179 and MW880198 from the ITS region and BLAST search in GenBank showed 100 and 98.11% alignment with previously published sequences of F. equiseti with accessions OM992323.1and MT558569.1 respectively. Accession number OM867571from the ß-tubulin region showed 100% sequence similarity with F. equiseti with accession MN653163.1. For pathogenicity, macroconidia from 2-week-old cultures on CLA medium were harvested to prepare spore suspension (1 × 106 conidia/ml). Koch's postulates were confirmed on nine bitter gourd plants (cv. Seminis-200) by applying spore suspension of fungal inoculum at 3-4 leaf stage separately on leaves by automizer, on collar region after making incision spore suspension was applied and in the root zone, 20ml spore suspension was added whereas distilled water was used as a control with three replications. Plants were kept under controlled conditions in the greenhouse with 65% to 75% humidity and the temperature was maintained at 32±2 °C for one week. After 7-8 days, inoculated plants began to exhibit symptoms of brown, necrotic leaf spots on the leaves of bitter gourd vines followed by yellowing of leaves that eventually turned brown. Roots showed slight discoloration and browning of vascular bundles and finally, the plants wilted after four weeks. while control plants remained symptomless. The symptoms resembled those noticed in the field. The fungus was re-isolated from leaves, collar region and roots, followed by morphological identification, and finally confirmed as F. equiseti. To the best of our knowledge, this is the first report of a leaf spot caused by F. equiseti in a bitter gourd from Pakistan. If the disease is not managed properly, it may cause a drastic effect on yield under favorable environmental conditions. The pathogen may also damage other cucurbitaceous crops cultivated in the area.

Corrigendum: Role of Promising Secondary Metabolites to Confer Resistance Against Environmental Stresses in Crop Plants: Current Scenario and Future Perspectives.

[This corrects the article DOI: 10.3389/fpls.2022.881032.].

Role of Promising Secondary Metabolites to Confer Resistance Against Environmental Stresses in Crop Plants: Current Scenario and Future Perspectives.

Plants often face incompatible growing environments like drought, salinity, cold, frost, and elevated temperatures that affect plant growth and development leading to low yield and, in worse circumstances, plant death. The arsenal of versatile compounds for plant consumption and structure is called metabolites, which allows them to develop strategies to stop enemies, fight pathogens, replace their competitors and go beyond environmental restraints. These elements are formed under particular abiotic stresses like flooding, heat, drought, cold, etc., and biotic stress such as a pathogenic attack, thus associated with survival strategy of plants. Stress responses of plants are vigorous and include multifaceted crosstalk between different levels of regulation, including regulation of metabolism and expression of genes for morphological and physiological adaptation. To date, many of these compounds and their biosynthetic pathways have been found in the plant kingdom. Metabolites like amino acids, phenolics, hormones, polyamines, compatible solutes, antioxidants, pathogen related proteins (PR proteins), etc. are crucial for growth, stress tolerance, and plant defense. This review focuses on promising metabolites involved in stress tolerance under severe conditions and events signaling the mediation of stress-induced metabolic changes are presented.

Evaluation of Bacterial Perpetuation Assays and Plant Biomolecules Antimicrobial Activity against Cotton Blight Bacterium Xanthomonas citri subsp. malvacearum; An Alternative Source for Food Production and Protection.

Cotton (Gossypium hirsutum) is a global cash crop which has gained importance in earning foreign exchange for each country. Bacterial blight caused by Xanthomonascitri subsp. malvacearum (Xcm) has been a seriousdisease in Pakistan's cotton belt on multiple occasions. Bacterium was isolated and identified through various biochemical and diagnostic tests wherehypersensitivity reaction, Gram staining, KOH (potassium hydroxide), catalase, starch hydrolysis, lecithinase and Tween 80 hydrolysis tests confirmed bacterium as Gram-negative and plant pathogenic. Xcm perpetuation assays wereevaluated on various cotton varieties under glasshouse conditions in completely randomized design by three different methods, wherein the scratch method proved to be the best upon CIM-496 and showed 83.33% disease incidence as compared with the other two methods, where Bt-3701 responded with 53.33% incidence via the spray gun method, and 50% with the water splash method on CIM-616, as compared with the control. Similarly, for disease severity percentage, Bt-3701 was pragmatic with 47.21% through scratch method, whereas, in the spray gun method, 45.51% disease severity was noted upon Bt-802, and 31.27% was calculated on Cyto-179 through the water splash method. Owing to the unique antibacterial properties of aqueous plant extracts, the poison food technique showed Aloe vera, Mentha piperita, Syzygiumcumini and Azadirachta indica with 17.77, 29.33, 18.33 and 20.22 bacterial colonies counted on nutrient agarmedium petri plate, respectively, as compared with the control. Measurement of the inhibition zone by disk diffusion technique showed Mentha piperita, Syzygiumcumini, Citrus limon, Moringa oleifera and Syzygium aromaticum to present the most promising results by calculating the maximum diameter of the inhibition zone, viz., 8.58, 8.55, 8.52, 8.49 and 8.41 (mm), respectively, at the highest tested concentration (75 ppm, parts per million) compared with the control. It is probable that the decoction's interaction with the pathogen population on the host plant will need to be considered in future experiments. However, at this moment, more research into the effective management of cotton bacterial blight by plant extracts in terms of concentration determination and development of biopesticides will provide future avenues to avoid environmental pollution.

Deploying Viruses against Phytobacteria: Potential Use of Phage Cocktails as a Multifaceted Approach to Combat Resistant Bacterial Plant Pathogens.

Plants in nature are under the persistent intimidation of severe microbial diseases, threatening a sustainable food production system. Plant-bacterial pathogens are a major concern in the contemporary era, resulting in reduced plant growth and productivity. Plant antibiotics and chemical-based bactericides have been extensively used to evade plant bacterial diseases. To counteract this pressure, bacteria have evolved an array of resistance mechanisms, including innate and adaptive immune systems. The emergence of resistant bacteria and detrimental consequences of antimicrobial compounds on the environment and human health, accentuates the development of an alternative disease evacuation strategy. The phage cocktail therapy is a multidimensional approach effectively employed for the biocontrol of diverse resistant bacterial infections without affecting the fauna and flora. Phages engage a diverse set of counter defense strategies to undermine wide-ranging anti-phage defense mechanisms of bacterial pathogens. Microbial ecology, evolution, and dynamics of the interactions between phage and plant-bacterial pathogens lead to the engineering of robust phage cocktail therapeutics for the mitigation of devastating phytobacterial diseases. In this review, we highlight the concrete and fundamental determinants in the development and application of phage cocktails and their underlying mechanism, combating resistant plant-bacterial pathogens. Additionally, we provide recent advances in the use of phage cocktail therapy against phytobacteria for the biocontrol of devastating plant diseases.

Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan.

Citrus viroid infection is emerging as a serious threat because of its efficient systemic movement within the host plant and its quick spread due to contaminated pruning tools. A survey was conducted to investigate the primary distribution and molecular characterization of Citrus bent leaf viroid (CBLVd) and its variants in different citrus cultivars. A total of 154 symptomatic citrus samples were collected and detected by RT‒PCR with newly designed specific primers with the incidence of 36.33%. During biological indexing study on Etrog citron, expressions of reduced leaf size, yellowing with a light green pattern, and bending were observed. Amplified products were sequenced and analyzed using a nucleotide BLAST search, which showed 98% homology with other CBLVd isolates. The results of the phylogenetic tree analysis showed the presence of two main groups (A and B), with the predominant variants of CBLVd, i.e., CVd-I-LSS (Citrus viroid Low Sequence Similarity) sequences, clustering in subgroup A1 along with newly detected CVd-I-LSS from Palestinian sweet lime (Citrus limettioides), which has been identified as a new host of CVd-I-LSS in Pakistan. Further analysis of the sequences in subgroup A1 showed that the variant of CVd-I-LSS infecting citrus cultivars had a close relationship with isolates reported from China, Japan, and Iran, which may have resulted from the exchange of planting material. This study also unveiled the variability in nucleotide sequences of CBLVd, which made it unable to be detected by old primers. The results of this study indicate that the widespread presence of divergent variants of CBLVd is a major concern for the citrus industry in Pakistan and other countries where virulent isolates of CBLVd are prevalent. These findings suggest the need for future research on effective management and quarantine measures to stop the spread of CBLVd.

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection.

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Effect of different combinations of antibiotics on fruit quality and antioxidant defense system in Huanglongbing infected Kinnow orchards.

Huanglongbing (HLB), also known as citrus greening disease, is the most devastating disease of citrus across the world, caused by the phloem limited fastidious bacterium 'Candidatus Liberibacter spp.'. This research was conducted on HLB infected 10-year-old Kinnow orchard located at Multan, Pakistan. Different classes of antibiotics in various combinations were applied on HLB-infected trees. The antibiotic treatments were applied before flowering in February, during fruit setting in April and at fruit growth stage in June. The different antibiotics combinations used were Ampicillin sodium + Rifampicin, Cefalexin + Rifampicin, Ampicillin sodium + Cefalexin, Ampicillin sodium + Cefalexin + Rifampicin and Control (distilled water). Different fruit qualitative and quantitative attributes were examined. The application of antibiotics significantly decreased 2-11% in flower, June and pre-harvest drops as compared to control. Further, antibiotics increased fruit weight and yield by five times while the juice content, total soluble solids, ripening index, total sugars, phenolic and vitamin C content were also increased in fruits. In addition, total soluble proteins, peroxidase and catalase activities were increased in fruits harvested from antibiotic treated plants compared to control, however the superoxidase dismutase activity was decreased in fruits of antibiotic treated plants. Finally, it is concluded that application of different antibiotics combinations helps in improving the fruit yield and different quality attributes of HLB infected Kinnow trees.

Antifungal Potential of Indigenous Medicinal Plants against Myrothecium Leaf Spot of Bitter Gourd ( Momordica charantia L.)

ABSTRACT Bitter gourd is of great importance due to its usage against the treatment of numerous ailments in human beings. A comprehensive survey at four localities of Southern Punjab, Pakistan was carried out to determine the severity of Myrothecium leaf spot. Maximum disease severity was at C1 (Chak 11/NP) and least at C2 (Kot Mehtab). Among isolated species Myrothecium roridum was found more prevalent and pathogenic as compared to M. verrucaria. Antifungal activity using solvent extracts of five medicinal plants (Mangifera indica, Melia azedarach, Nicotiana tabacum, Moringa oleifera and Eucalyptus globosum) were evaluated against isolated species by agar well diffusion method at various concentrations (0.01, 0.10, 1.0 and 10.0 µg / mL). N. tabacum revealed maximum zone size (13.40 mm and 8.28 mm) with ethanol and chloroform solvents respectively followed by M. azedarach (9.00mm and 6.48mm). However, least inhibition was observed with ethanol and chloroform extracts of E. globosum (6.04mm and 3.88mm zone size respectively). Ethanol extracts showed highest activity when compared to chloroform extracts. Qualitative phytochemical analysis showed that all the selected plants are rich in chemical compounds such as alkaloids, terpenoids, flavonoids and phenols whereas Saponins was only present in N. tabacum while absent in rest of the extracts.