Tuning active sites on biochars for remediation of mercury-contaminated soil: A comprehensive review.

Mercury (Hg) contamination is acknowledged as a global issue and has generated concerns globally due to its toxicity and persistence. Tunable surface-active sites (SASs) are one of the key features of efficient BCs for Hg remediation, and detailed documentation of their interactions with metal ions in soil medium is essential to support the applications of functionalized BC for Hg remediation. Although a specific active site exhibits identical behavior during the adsorption process, a systematic documentation of their syntheses and interactions with various metal ions in soil medium is crucial to promote the applications of functionalized biochars in Hg remediation. Hence, we summarized the BC's impact on Hg mobility in soils and discussed the potential mechanisms and role of various SASs of BC for Hg remediation, including oxygen-, nitrogen-, sulfur-, and X (chlorine, bromine, iodine)- functional groups (FGs), surface area, pores and pH. The review also categorized synthesis routes to introduce oxygen, nitrogen, and sulfur to BC surfaces to enhance their Hg adsorptive properties. Last but not the least, the direct mechanisms (e.g., Hg- BC binding) and indirect mechanisms (i.e., BC has a significant impact on the cycling of sulfur and thus the Hg-soil binding) that can be used to explain the adverse effects of BC on plants and microorganisms, as well as other related consequences and risk reduction strategies were highlighted. The future perspective will focus on functional BC for multiple heavy metal remediation and other potential applications; hence, future work should focus on designing intelligent/artificial BC for multiple purposes.

Effect of green-synthesized copper oxide nanoparticles on growth, physiology, nutrient uptake, and cadmium accumulation in Triticum aestivum (L.).

Cadmium (Cd) stress in crops has been serious concern while little is known about the copper oxide nanoparticles (CuO NPs) effects on Cd accumulation by crops. This study investigated the effectiveness of CuO NPs in mitigating Cd contamination in wheat (Triticum aestivum L.) cultivation through a pot experiment, presenting an eco-friendly solution to a critical agricultural concern. The CuO NPs, synthesized using green methods, exhibited a circular shape with a crystalline structure and a particle size ranging from 8 to 12 nm. The foliar spray of CuO NPs was applied in four different concentrations i.e. control, 25, 50, 75, 100 mg/L. The obtained data demonstrated that, in comparison to the control group, CuO NPs had a beneficial influence on various growth metrics and straw and grain yields of T. aestivum. The green CuO NPs improved T. aestivum growth and physiology under Cd stress, enhanced selected enzyme activities, reduced oxidative stress, and decreased malondialdehyde levels in the T. aestivum plants. CuO NPs lowered Cd contents in T. aestivum tissues and boosted the uptake of essential nutrients from the soil. Overall, foliar applied CuO NPs were effective in minimizing Cd contents in grains thereby reducing the health risks associated with Cd excess in humans. However, more in depth studies with several plant species and application methods of CuO NPs are required for better utilization of NPs in agricultural purposes.

Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 promote plant growth through upregulation of growth-promoting genes in upland cotton.

AIMS: The potential of endophytic Bacillus strains to improve plant growth and yield was evaluated. METHODS AND RESULTS: Endophytic Bacillus altitudinis HNH7 and Bacillus velezensis HNH9 were evaluated for their growth-promoting traits. In an in vitro plate assay, HNH7 and HNH9 exhibited proteolytic, amylolytic, lipolytic and cellulolytic activity. HNH7 and HNH9 were able to solubilize iron by producing siderophores but were unable to solubilize insoluble phosphate. PCR confirmed the presence of four growth-promoting genes viz. pvd, budA, asbA and satA in the genome of HNH7, while HNH9 also possessed the same genes except for budA. In a greenhouse experiment, HNH7 and HNH9 promoted the growth of upland cotton plants by upregulating the expression of growth-linked genes, EXP6, ARF1, ARF18, IAA9, CKX6 and GID1b. However, the expression of genes involved in ethylene biosynthesis, that is ERF and ERF17 was downregulated after treating the plants with HNH7 and HNH9 compared to the control. Furthermore, cotton plants treated with HNH7 and HNH9 exhibited a significantly higher rate of photosynthesis and stomatal conductance. CONCLUSION: HNH7 and HNH9 showed a promising potential to promote the growth of cotton plants. SIGNIFICANCE AND IMPACT OF STUDY: Research on plant growth-promoting Bacillus strains can lead to the formation of biofertilizers.

First report of leaf blight disease of Livistona chinensis caused by Alternaria alternata in Pakistan.

Livistona chinensis (Jacq.) is also known as fan palm and is commonly grown in the subtropical region of the world. This plant is widely cultivated in Asia for ornamental purpose and also used in Chinese medicines (Li et al. 2019). In May 2021, severe leaf blight was observed on L. chinensis leaves in ornamental plant nurseries, located at Pattoki (30°59'41.5"N 73°48'43.8"E) District Kasur, Punjab province, Pakistan. The disease incidence was up to 50% and the initial symptoms appeared as chlorotic brown spots on the upper portion of leaves. Later, the spots expanded and changed into elliptical lesions on the leaves. The lesions with dark brown margins coalesced to cause extensive tissue necrosis of leaves and exhibited blight (Figure 1). Two to three leaves were taken from each infected plant. Infected leaves of each sample of L. chinensis were excised into small pieces (3-4 mm) with the help of sterilized scissor and surface disinfected with 1% NaClO for 20s and rinsed 3 times with sterilized distilled water. To isolate the potential causal organism, these pieces were plated on Potato Dextrose Agar (PDA) medium and incubated at 28 °C with 70 % relative humidity for 7 days. Purified cultures were obtained through single spore culture on PDA. All obtained isolates were preserved in 30% glycerol at -80°C. The fungal colony colour was olive to dark greenish and dark brown to black on the reverse side. The conidia (n=36 per isolate) were greenish to brown in colour, ellipsoid to obclavate, ovoid, irregular and measured an average range from 10.9 to 30.7 µm long x 6.3 to 12.5 µm wide with 2 to 5 transverse and 0 to 3 longitudinal septa (Figure 2). The genomic DNA was extracted from all isolates (n=40) and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, Alt a1 major allergen (ALT) gene, glyceraldehyde-3-phosphate dehydrogenase (GPD), actin (ACT) gene and histone 3 genes were amplified using ITS1/4 (White et al. 1990), Alt-4for/Alt-4rev (Lawrence et al. 2013), GPD1/GPD2 (Guerber et al. 2003), ACT512F/ACT783R (Carbone and Kohn, 1999) and H3-1a/H3-1b (Luan et al. 2007). Based on morphological and molecular characteristics, all isolates were identified as Alternaria alternata. The sequences of the representative isolate APLB-3 were submitted in the GenBank with the accession numbers (ITS: MZ663802), (ALT: MZ666883), (ACT: MZ666885), (GPD: MZ666884), and (Histone3: MZ666886) showing 100% similarity with ITS accession MK968038, ALT accession MN702781, ACT accession MT318253, GPD accession MT524743 and histone 3 accession MH824369. For pathogenicity test, potted L. chinensis plants (n=9) leaves were pin-pricked with sterilized needle (Bajwa et al. 2010) and inoculated with spore suspensions (107 spore/ml) of APLB-3 (1ml/leaf) to confirm Koch's postulates. After 14 days, the inoculated leaves showed chlorotic brown spots and leaf blight symptoms similar to those observed on infected plants in nurseries. The plants grown as the control group (n=9) were sprayed with sterilized distilled water and had no symptoms (Figure 3). The experiment was performed three times. The fungal pathogen was re-isolated from the artificial inoculated leaf tissues and identified as A. alternata based on morphological and molecular characterization. To our knowledge, this is the first record of A. alternata causing leaf blight disease of L. chinensis in Pakistan. This disease may potentially decrease the value of ornamental plants in Pakistan under favourable conditions and proper management strategies should be applied.

First record of Leaf Spot of Ficus religiosa Caused by Alternaria alternata in Pakistan.

Ficus religiosa (L.) belongs to the family Moraceae, native to India and commonly known as 'Peepal'. It has high medicinal value due to its antibacterial, antiviral and antioxidant properties (Singh et al., 2015; Kalpana et al., 2009). In August 2021, leaf spots were observed on F. religiosa trees in Pabbi forest park Kharian (32°50'01.4"N 73°50'17.7"E), District Gujrat, Pakistan. The disease incidence was recorded approximately 30%. The leaf spots were irregular in shape, brown in colour, 3-9 mm in size and encircled by yellowish halo. In severe condition, the spots coalesced and produced necrotic areas on leaf surface (Figure 1). The samples (n=21) were collected based on symptoms and infected leaf segments were excised into small pieces, surface disinfected with 1% NaClO for 20s and rinsed 3 times with sterilized distilled water. The pieces were plated on Potato Dextrose Agar (PDA) medium and incubated at 28°C for 7 days. All the pure cultures were obtained through single spore method on PDA and preserved in 30% glycerol at -80°C. The colonies were olive green to dark brown with white margin and later turned dark olive or black with enormous sporulation. Conidia (n=24) were obclavate, ovoid, brown in colour and measuring 10.2 to 34.1 µm long x 5.9 to 12.3 µm wide with 1 to 6 transverse and 1 to 3 longitudinal septa (Figure 2). Based on these characteristics, the pathogen was identified as Alternaria alternata (Gilardi, G., et al. 2019). For molecular identification, the Internal Transcribed Spacers (ITS) region, endopolygalacturonase (endoPG) gene and major allergen (Alt a1) gene were amplified using ITS1/4 (White et al. 1990), PG3/PG2b (Andrew et al. 2009) and Alt-4for/Alt-4rev (Lawrence et al. 2013) primers respectively. Based on molecular characteristics, all isolates were identified as A. alternata. The sequences of the representative isolate FLB-1 were submitted in the GenBank with the accession numbers OL514181 for ITS, OK315658 for endoPG and OK315659 for Alt al showing 100% similarity with ITS accession KP124298, and endoPG accession AY205020 and 99.7% with Alt al accession KP123847 sequences of CBS106.24 A. alternata after BLASTn queries. The Phylogenetic reconstruction based on maximum likelihood, using Mega X (Kumar et al. 2018) and FLB-1 grouped with A. alternata (Figure 3). Pathogenicity test was performed on nine months old healthy F. religiosa (L.) seedlings (n=12) to fulfil the Koch's postulates. The leaves were pinpricked and sprayed with FLB-1 conidial suspension (107 spores/ml) by using atomizer (Bajwa et al., 2010). The leaves of F. religiosa (L.) seedlings (n=12) sprayed with sterilized distilled water served as control. All the seedlings were incubated at 25 ± 3°C in the glasshouse. The experiment was performed three times under the same conditions. The typical symptoms appeared on inoculated leaves after 7-14 days that were similar to the symptoms observed on original infected F. religiosa (L.) trees. In the control treatment leaves remained asymptomatic (Figure 4). The pathogen from the artificial infected leaves was re-isolated and identified as A. alternata based on morphological and molecular characteristics. To our knowledge, this is the first report of leaf spot of F. religiosa (L.) caused by A. alternata in Pakistan. The leaves of F. religiosa (L.) are commonly used in Asia for different purposes and this leaf spot disease may represent a significant threat to F. religiosa (L.) tree health.

First Report of Bacterial Leaf Spot of Ficus benghalensis Caused by Pseudomonas cichorii in Pakistan.

Ficus benghalensis L. belongs to the family Moraceae, native to Asia and commonly known as Banyan. It has been identified as an important medicinal tree due to its antioxidant, anti-diabetic, and anti-inflammatory properties (Singh et al., 2009; Thite et al., 2014). In March 2021, leaf spots were observed on Banyan trees in the Kharian forest zone, District Gujrat, Punjab Province, Pakistan. Initial symptoms on leaves were irregular, water-soaked, and light brown lesions. The lesions turned dark brown at the centre, and the margins gradually turned yellow. The average size of lesions was 12 to 20 × 8 to 13 mm. The lesions coalesced and produced necrotic areas on the leaf (Figure 1). Samples (n=34) were collected based on symptoms and infected leaf segments were excised into small pieces (10-20 mm), surface disinfected with 1% NaClO for 10 seconds and rinsed three times with sterilized distilled water (SDW). Ten pieces/sample were mashed and soaked in 1.5 ml SDW to obtain a suspension. Later, 10 µL of the suspension was streaked on Nutrient agar (NA) and King's B medium (KBM) and incubated for 72 h at 30°C. After 72 h bacterial colonies appeared on NA and KBM medium. Each colony was re-streaked for three times to obtain the purified colonies. Morphological and biochemical characteristics of isolated bacterial cultures were performed by following the method of Schaad et al. (2001). Bacterial colonies appeared pale yellow to creamy, smooth, and circular with undulated margins on both NA and KBM medium. The colonies produced a fluorescent blue colour on KBM under the UV light. Isolated bacterial cultures were positive for oxidase, negative for levan production and arginine dihydrolase. Based on these characteristics, the pathogen was identified as Pseudomonas species. For molecular identification, 16S rRNA and rpoB genes were amplified and sequenced using the following primers: 27F/1492R (Lane, 1991) and LAPS/LAPS27 (Ait Tayeb et al. 2005), respectively. All the isolates were identified as P. cichorii after BLASTn analysis. The sequences of isolate BLS-01 obtained in this study were deposited in GenBank with accession No. OK397593 for 16S rRNA and OK423684 for rpoB exhibiting 100 % similarity with P. cichorii Accession No. MK356431 for 16S rRNA and JQ267563 for rpoB. A pathogenicity test was performed on healthy Banyan seedlings to fulfil Koch's postulates. Leaves from seedling plants were inoculated with 3 mL of BLS-01 suspension (108 CFU/ml) by spraying the inoculum on leaves using a sterilized spray bottle. The leaves sprayed with sterilized distilled water served as control (Figure 2). The experiment was performed three times following the same protocol as described above. Symptoms that appeared on inoculated leaves after 7-10 days were similar to the symptoms observed on original infected Banyan tree leaves in the forest zone. Control leaves remained asymptomatic during the whole experiment. The pathogen from the artificial infected leaves was re-isolated and identified as P. cichorii based on morphological, biochemical, and molecular characteristics. To our knowledge, this is the first report of leaf spot of F. benghalensis caused by P. cichorii in Pakistan.

First record of Colletotrichum alienum Causing postharvest Anthracnose disease of mango fruit in China.

Mango (Mangifera indica L.) is one of the world's most significant economic fruit crops, and China is the second-largest producer of mango (Kuhn et al., 2017). Postharvest mango anthracnose is caused by Colletotrichum species and reduce the self-life of mature fruit (Wu et al., 2020). Colletotrichum species also cause postharvest anthracnose and fruit rot disease of Apple, Banana and Avocado (Khodadadi et al., 2020; Vieira et al., 2017; Sharma et al., 2017). In July 2019, mango fruits cv. 'Jin-Hwang' were observed at different fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district, Beijing, China, exhibiting typical symptoms of anthracnose including brown to black lesions in different size (≤ 2 cm) with identified border on the mango fruit surface. Later, the lesions were coalesced and extensively cover the surface area of the fruit. The lesions were also restricted to peel the fruit and pathogen invaded in the fruit pulp. About 30% of mango fruits were affected by anthracnose disease. The margins of lesions from infected mango fruits (n=56) were cut into 2 × 2 mm pieces, surface disinfected with NaClO (2% v/v) for 30 s, rinsed thrice with distilled water for 60s. These pieces were placed on PDA medium and incubated at 25°C for 7 days. Pure culture of fungal isolates was obtained by single spore isolation technique. Initially, the fungal colony was off white, and colony extended with time, turning light gray at the center. The morphological examination revealed that conidia were hyaline, oblong, and unicellular. The conidia were measured from 10 days old culture and dimensions varied from 13.3 to 15.8 µm in length and 4.6 to 6.1 µm in width. For molecular identification, a multi-locus sequence analysis; the Internal Transcribed Spacers (ITS) region, partial actin (ACT) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and chitin synthase (CHS-1) gene were amplified by using the primer sets ITS1/4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF1/GDR1 (Guerber et al. 2003) and CHS1-79F/CHS-1-354R (Carbone and Kohn 1999) respectively. The partial sequences of MTY21 were deposited to GenBank accessions (MT921666 (ITS), MT936119 (ACT), MT936120 (GAPDH) and MT936118 (CHS-1). All obtained sequences showed 100% similarity with reported sequences of Colletotrichum alienum ICMP.18691 with accessions numbers JX010217 (ITS), JX009580 (ACT), JX010018 (GAPDH) and JX009754 (CHS-1) which represented the isolate MTY21 identified as C. alienum by constructing Maximum Likelihood phylogenetic tree analysis using Mega X (Kumar et al., 2018). For the confirmation of Koch's postulates, the pathogenicity test was conducted on 36 fresh healthy mango fruits for each treatment. Fruits were punctured with the help of a sterilized needle to create 2mm2 wounds and inoculated with 10µL inoculum (107 spores/mL) of MTY21. Control mango fruits were inoculated with 10µL sterilized distilled water and incubated at 25 °C with 90% relative humidity. The lesions appeared at the point of inoculation and gradually spread on the fruit surface after 7 days post inoculation. The symptoms were similar to the symptoms on original fruit specimens. The re-isolated fungus was identified as C. alienum based on morphological and molecular analysis. Mango anthracnose disease caused by several Colletotrichum species has been reported previously on mango in China (Li et al., 2019). Liu et al. (2020) reported C. alienum as the causal organism of anthracnose disease on Aquilaria sinensis in China. C. alienum has been previously reported causing mango anthracnose disease in Mexico (Tovar-Pedraza et al., 2020) To our knowledge, this is the first report of C. alienum causing postharvest anthracnose of mango in China. The prevalence of C. alienum was 30% on mango fruit which reflects the importance of this pathogen as a potential problem of mango fruit in China.

First report of post-harvest Fusarium rot of mandarin Citrus reticulata cv. 'Kinnow' caused by Fusarium equiseti in Pakistan.

Citrus reticulata cv. 'Kinnow' mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O'Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 µL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 µL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.

Transcriptional Profiling of Diffusible Lipopeptides and Fungal Virulence Genes During Bacillus amyloliquefaciens EZ1509-Mediated Suppression of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that infects multiple crops, and its control is an unremitting challenge. In this work, we attempted to gain insights into the pivotal role of lipopeptides (LPs) in the antifungal activity of Bacillus amyloliquefaciens EZ1509. In a comparative study involving five Bacillus strains, B. amyloliquefaciens EZ1509 harboring four LPs biosynthetic genes (viz. surfactin, iturin, fengycin, and bacilysin) exhibited promising antifungal activity against S. sclerotiorum in a dual-culture assay. Our data demonstrated a remarkable upsurge in LPs biosynthetic gene expression through quantitative reverse transcription PCR during in vitro interaction assay with S. sclerotiorum. Maximum upregulation in LPs biosynthetic genes was observed on the second and third days of in vitro interaction, with iturin and fengycin being the highly expressed genes. Subsequently, Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis confirmed the presence of LPs in the inhibition zone. Scanning electron microscope analysis showed disintegration, shrinkage, plasmolysis, and breakdown of fungal hyphae. During in planta evaluation, S. sclerotiorum previously challenged with EZ1509 showed significant suppression in pathogenicity on detached leaves of tobacco and rapeseed. The oxalic acid synthesis was also significantly reduced in S. sclerotiorum previously confronted with antagonistic bacterium. The expression of major virulence genes of S. sclerotiorum, including endopolygalacturonase-3, oxalic acid hydrolase, and endopolygalacturonase-6, was significantly downregulated during in vitro confrontation with EZ1509.

First Report of Pre-Harvest Soft Rot of Peach Fruit (Prunus persica) Caused by Enterobacter mori in China.

Peach (Prunus persica L. Batsch) is one of the major fruit crops of China and a rich source of vital nutrients and fibers. In July 2019, a peach orchard was visited at Pinggu District, Beijing, China. The average temperature and relative humidity at the sampling site were 28±2 °C and 75±2 % respectively. In the orchard, unripe and near to ripe peach fruits cv. 'China Pearl' were observed with soft brown patches (3-4×2-3cm) on the surface. The samples (n=25) were collected based on typical symptoms. When the rotten part of the fruit was pressed, a liquid oozed out of the fruit emitting an unpleasant odor. The disease incidence of fruit soft rot was 22 %. The percent incidence was calculated based on total number of infected fruits divided by total number of fruits examined. Infected fruit tissues were excised into small pieces (5mm) and surface disinfested with 1% NaClO for 25s and rinsed twice with sterilized distilled water (SDW). These pieces (4-5 pieces per sample) were macerated with 1.5 ml SDW in a 2 ml tube. Later, 5 µl supernatant was streaked on Luria Bertani (LB) agar plates and incubated at 28 ºC for 3 days. Oozing liquid (50 µl) was also inoculated on 100ml LB broth and incubated for 24 h and 5 µl was streaked on LB agar medium and incubated at 28 ºC. Purified colonies were obtained by re-streaking 3 times. The isolate formed creamy to light yellowish, irregular, round, rough or smooth-looking colonies on LB medium and pink colonies on Eosin Methylene blue. The bacterium was rod shaped, measuring 0.8 to 1.4 µm in length and 0.2 to 0.5 µm in width (Fig 1) observed using a scanning electron microscope (Hitachi-SU8010). Morphological characteristics were similar to previously described characteristics of Enterobacter spp. (Zhu et al. 2011; Nishijima et al. 1987). Genomic DNA was extracted from all twenty-five isolates by using TIANamp Bacteria DNA Kit (Tiangen-Biotech, Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene of 25 isolates was amplified by using universal primers (27F:5'-AGAGTTTGATCCTGGCTCAG-3',/1492R:5'-CTACGGCTACCTTGTTACGA-3'), and rpoB gene (F2: 5'-AACCAGTTCCGCGTTGGCCTGG-3', R2: 5'-CCTGAACAACACGCTCGGA-3') (Mollet et al., 1997). Sequences for EPT-1 were submitted to GenBank (accessions MN548761 (16S rRNA), MN594495 (rpoB)). Accessions MN548761 and MN594495 had 99.35 and 99.77% sequence identity with E. mori (GenBank accessions KF747680, GQ406571). Maximum Likelihood phylogenetic tree constructed (1000 replicates) using Mega X (Kumar et al. 2018) indicated that isolate EPT-1 clustered with E. mori sequences. To confirm the pathogenicity, medium-sized (n=60) surface disinfested ripe peach fruits cv. 'China Pearl' were wound inoculated with 5 µl suspension of EPT-1 (107cfu/ml) and the control fruit were treated with 5 µl sterilized water. The fruit were kept in sterilized plastic box and incubated at 28 ºC for 7 days at 70% relative humidity, and the pathogenicity test was repeated 3 times. Symptoms similar to the original fruit samples were observed on all inoculated fruit (Fig 2). The pathogen was re-isolated, the colonies obtained from the oozing liquid were similar to those of infected fruit tissues and identified as E. mori based on morphological and sequencing analysis. Previously, E. mori has been reported to cause bacterial wilt on Morus alba L. in China (Zhu et al. 2011). To our knowledge, this is the first report of E. mori causing soft rot of peaches in China.

Occurrence and molecular characterization of Meloidogyne graminicola on rice in Central Punjab, Pakistan.

Meloidogyne graminicola threatens global rice production, yet is understudied for many areas where it is cultivated. To better understand the prevalence and incidence of M. graminicola in central Punjab, Pakistan, we carried out field surveys of rice fields in the districts of Faisalabad and Chiniot. M. graminicola isolates were recovered from soil and root samples and identified on the basis of perineal patterns and rDNA ITS-based sequencing. The severity of nematode attack on rice roots and infested fields at various locations was based on galling index, root-knot nematode juveniles per root system, juveniles per 100 ml of soil, and prevalence of stylet-bearing nematodes and non-stylet-bearing nematodes. Maximum prevalence (22.5 and 27.5%) and minimum prevalence (17.5 and 20%) of M. graminicola was observed in Chiniot and Faisalabad, respectively. Eleven alternate host-plant species were examined in this study revealing varying degrees of M. graminicola infestation. ITS sequencing and phylogenetic analysis indicated that isolates from this study form a well-resolved clade with others from Asia, while another isolate falls outside of this clade in an unresolved polytomy with those from Europe and South America. Though monophyletic with the other M. graminicola, the isolates from Pakistan are distinguished by their high genetic variability and long branch lengths relative to the other isolates of M. graminicola, suggesting Pakistan as a possible ancestral area. Our results indicate that rice is severely attacked by a genetically diverse and aggressive M. graminicola, necessitating the development of appropriate control measures for its management in rice and other graminaceous crops.

Melatonin induces proline, secondary metabolites, sugars and antioxidants activity to regulate oxidative stress and ROS scavenging in salt stressed sword lily.

Sword lily is regarded as a useful and commercially demanding cut flower crop; hence, assessing its responses to abiotic stress, particularly salt stress, is vital. Melatonin (MT) exhibits stress tolerance in crop plants and is an emerging stress relieving alternative to chemicals. Nevertheless, the possible process underlying the effects of MT under salt stress has yet to be fully elucidated in plants. Herein, the salt stress (SS) mitigation potential of MT was assessed in a commercially important cut flower, sword lily. Melatonin, expressed as MT1, MT2, MT3, and MT4, was administered at concentrations of 0.2, 0.4, 0.6, and 0.8 mM. The results revealed that SS (5 dS m-1) restricted the growth and physiological aspects of sword lily. Furthermore, malondialdehyde (MDA), hydrogen peroxide (H2O2), membrane permeability, endogenous proline, and soluble protein contents were enhanced in SS. MT application improved morphological traits, photosynthetic pigments, and corm traits. The application of MT mitigated the effects of SS stress in Gladiolus grandiflorus plants by improving growth and photosynthetic pigments. MT application under SS improved the reducing and non-reducing sugar and NPK contents of the sword lily. Furthermore, MT improved the levels of secondary metabolites, such as anthocyanins, flavonoids, and ascorbic acid, in sword lily. Moreover, MT supplementation ameliorated salt-induced oxidative stress in the gladiolus, as depicted by a decrease in stress markers (EL, MDA, and H2O2) and an increase in defense-related enzymes (POD, CAT, and SOD) with highest increase in the MT3 treatment under salinity stress. The SOD and CAT enzyme activities were 3-3.6-fold higher in the MT3 under stress than the control. In conclusion, MT applications on cut flowers can be an effective strategy to reduce salt stress and can be used to regulate salinity stress in cut flower production. MT can be used as a safe alternative to other agrochemicals to maintain the growth and flower quality of sword lilies, with beneficial effects during vase life.

Synergistic interplay between melatonin and hydrogen sulfide enhances cadmium-induced oxidative stress resistance in stock (Matthiola incana L.).

Ornamental crops particularly cut flowers are considered sensitive to heavy metals (HMs) induced oxidative stress condition. Melatonin (MLT) is a versatile phytohormone with the ability to mitigate abiotic stresses induced oxidative stress in plants. Similarly, signaling molecules such as hydrogen sulfide (H2S) have emerged as potential options for resolving HMs related problems in plants. The mechanisms underlying the combined application of MLT and H2S are not yet explored. Therefore, we evaluated the ability of individual and combined applications of MLT (100 µM) and H2S in the form of sodium hydrosulfide (NaHS), a donor of H2S, (1.5 mM) to alleviate cadmium (Cd) stress (50 mg L-1) in stock (Matthiola incana L.) plants by measuring various morpho-physiological and biochemical characteristics. The results depicted that Cd-stress inhibited growth, photosynthesis and induced Cd-associated oxidative stress as depicted by excessive ROS accumulation. Combined application of MLT and H2S efficiently recovered all these attributes. Furthermore, Cd stress-induced oxidative stress markers including electrolyte leakage, malondialdehyde, and hydrogen peroxide are partially reversed in Cd-stressed plants by MLT and H2S application. This might be attributed to MLT or H2S induced antioxidant plant defense activities, which effectively reduce the severity of oxidative stress indicators. Overall, MLT and H2S supplementation, favorably regulated Cd tolerance in stock; yet, the combined use had a greater effect on Cd tolerance than the independent application.

Biostimulants: A sufficiently effective tool for sustainable agriculture in the era of climate change?

Climate change is currently considered as one of the main concerns of the agriculture sector, as it limits crop production and quality. Furthermore, the current context of global crisis with international political instability and war conflicts over the world is pushing the agriculture sector even more to urgently boost productivity and yield and doing so in a sustainable way in the current frame of climate change. Biostimulants can be an effective tool in alleviating the negative effects of environmental stresses to which plants are exposed, such as drought, salinity, heavy metals and extreme temperatures etc. Biostimulants act through multiple mechanisms, modifying gene expression, metabolism and phytohormone production, promoting the accumulation of compatible solutes and antioxidants and mitigating oxidative stress. However, it is important to keep in mind that the use and effect of biostimulants has limitations and must be accompanied by other techniques to ensure crop yield and quality in the current frame of climate change, such as proper crop management and the use of other sustainable resources. Here, we will not only highlight the potential use of biostimulants to face future agricultural challenges, but also take a critical look at their limitations, underlining the importance of a broad vision of sustainable agriculture in the context of climate change.

Amelioration of chromium toxicity in wheat plants through exogenous application of nano silicon.

Chromium (Cr) contamination in agricultural soils poses a risk to crop productivity and quality. Emerging nano-enabled strategies show great promise in remediating soils contaminated with heavy metals and enhancing crop production. The present study was aimed to investigate the efficacy of nano silicon (nSi) in promoting wheat growth and mitigating adverse effects of Cr-induced toxicity. Wheat seedlings exposed to Cr (K2Cr2O7) at a concentration of 100 mg kg-1 showed significant reductions in plant height (29.56%), fresh weight (35.60%), and dry weight (38.92%) along with enhanced Cr accumulation in roots and shoots as compared to the control plants. However, the application of nSi at a concentration of 150 mg kg-1 showcased substantial mitigation of Cr toxicity, leading to a decrease in Cr accumulation by 27.30% in roots and 35.46% in shoots of wheat seedlings. Moreover, nSi exhibited the capability to scavenge oxidative stressors, such as hydrogen peroxide (H2O2), and malondialdehyde (MDA) and electrolyte leakage, while significantly enhancing gas exchange parameters, total chlorophyll content, and antioxidant activities (enzymatic and nonenzymatic) in plants grown in Cr-contaminated soil. This study further found that the reduced Cr uptake by nSi application was due to downregulating the expression of HMs transporter genes (TaHMA2 and TaHMA3), alongwith upregulating the expression of antioxidant-responsive genes (TaSOD and TaSOD). The findings of this investigation highlight the remarkable potential of nSi in ameliorating Cr toxicity. This enhanced efficacy could be ascribed to the distinctive size and structure of nSi, which augment its ability to counteract Cr stress. Thus, the application of nSi could serve as a viable solution for production of crops in metal contaminated soils, offering an effective alternative to time-consuming and costly remediation techniques.

Endophytic Bacillus atrophaeus CHGP13 and salicylic acid inhibit blue mold of lemon by regulating defense enzymes.

Lemons (Citrus limon L.) are one of the most economically important and consumed fruit worldwide. The species is vulnerable to several postharvest decay pathogens, of which Penicillium italicum associated with blue mold disease is the most damaging. This study investigates the use of integrated management for blue mold of lemon using lipopeptides (LPs) extracted from endophytic Bacillus strains and resistance inducers. Two resistance inducers; salicylic acid (SA) and benzoic acid (BA) were tested at 2, 3, 4, and 5 mM concentrations against the development of blue mold on lemon fruit. The 5 mM SA treatment produced the lowest disease incidence (60%) and lesion diameter (1.4 cm) of blue mold on lemon fruit relative to the control. In an in vitro antagonism assay eighteen Bacillus strains were evaluated for their direct antifungal effect against P. italicum; CHGP13 and CHGP17 had the greatest inhibition zones of 2.30 and 2.14 cm. Lipopeptides (LPs) extracted from CHGP13 and CHGP17 also inhibited the colony growth of P. italicum. LPs extracted from CHGP13 and 5 mM SA were tested as single and combined treatments against disease incidence and lesion diameter of blue mold on lemon fruit. SA + CHGP13 + PI had the lowest disease incidence (30%) and lesion diameter (0.4 cm) of P. italicum on lemon fruit relative to the other treatments. Furthermore, the lemon fruit treated with SA + CHGP13 + PI had the highest PPO, POD, and PAL activities. The postharvest quality analysis of the lemon fruit including fruit firmness, total soluble solids, weight loss, titratable acidity, and ascorbic acid content revealed that the treatment SA + CHGP13 + PI had little effect on fruit quality compared to the healthy control. These findings indicate that Bacillus strains and resistance inducers can be used as components of integrated disease management for the blue mold of lemon.

Biocontrol potential of lipopeptides produced by the novel Bacillus subtilis strain Y17B against postharvest Alternaria fruit rot of cherry.

The use of synthetic fungicides against postharvest Alternaria rot adversely affects human health and the environment. In this study, as a safe alternative to fungicides, Bacillus subtilis strain Y17B isolated from soil exhibited significant antifungal activity against Alternaria alternata. Y17B was identified as B. subtilis based on phenotypic identification and 16S rRNA sequence analysis. To reveal the antimicrobial activity of this strain, a PCR-based study detected the presence of antifungal lipopeptide (LP) biosynthetic genes from genomic DNA. UPLC Q TOF mass spectrometry analysis detected the LPs surfactin (m/z 994.64, 1022.68, and 1026.62), iturin (m/z 1043.56), and fengycin (m/z 1491.85) in the extracted LP crude of B. subtilis Y17B. In vitro antagonistic study demonstrated the efficiency of LPs in inhibiting A. alternata growth. Microscopy (SEM and TEM) studies showed the alteration of the morphology of A. alternata in the interaction with LPs. In vivo test results revealed the efficiency of LPs in reducing the growth of the A. alternata pathogen. The overall results highlight the biocontrol potential of LPs produced by B. subtilis Y17B as an effective biological control agent against A. alternata fruit rot of cherry.

Preharvest melatonin foliar treatments enhance postharvest longevity of cut tuberose via altering physio-biochemical traits.

Introduction: Melatonin (MLT) is a bioactive molecule involved in the physiological functioning of plants. Reports related to preharvest applications of melatonin on the postharvest performance of cut flowers are not available in the literature. Materials & methods: This study evaluated the effects of different concentrations of exogenous MLT [0 mM (MT0), 0.5 mM (MT1), 0.7 mM (MT2), 1 mM (MT3)] applied preharvest on the physiological characteristics and postharvest performance of cut tuberose, a globally demanded cut flower. Results & discussion: The results revealed that all treatments increased postharvest vase life by up to 4 d. The MT1, MT2, and MT3 treatments increased total soluble proteins (TSP) by 25%, 41%, and 17%, soluble sugars (SS) by 21%, 36%, and 33%, an+d postharvest catalase (CAT) activity by 52%, 66%, and 70%, respectively. Malondialdehyde (MDA) and hydrogen peroxide (H2O2) decreased in all preharvest treatments by up to 23% and 56%, respectively. Proline concentration decreased in all treatments, particularly MT3 (38%). These findings suggest that preharvest MLT treatment is a promising strategy for improving the postharvest quality of cut tuberose.

Bacillus thuringiensis CHGP12 uses a multifaceted approach for the suppression of Fusarium oxysporum f. sp. ciceris and to enhance the biomass of chickpea plants.

BACKGROUND: Bacillus species synthesize antifungal lipopeptides (LPs) making them a sustainable and eco-friendly management option to combat Fusarium wilt of chickpea. RESULTS: In this study, 18 endophytic Bacillus strains were assessed for their antifungal activity against Fusarium oxysporum f. sp. ciceris (FOC) associated with Fusarium wilt of chickpea. Among them, 13 strains produced significant inhibition zones in a direct antifungal assay while five strains failed to produce the inhibition of FOC. Bacillus thuringiensis CHGP12 exhibited the highest inhibition 3.45 cm of FOC. The LPs extracted from CHGP12 showed significant inhibition of the pathogen. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that CHGP12 possessed the ability to produce fengycin, surfactin, iturin, bacillaene, bacillibactin, plantazolicin, and bacilysin. In an in vitro qualitative assay CHGP12 exhibited the ability to produce lipase, amylase, cellulase, protease, siderophores, and indole 3-acetic acid (IAA). IAA and gibberellic acid (GA) were quantified using ultra-performance liquid chromatography (UPLC) with 370 and 770 ng mL-1 concentrations of IAA and GA respectively. Furthermore, the disease severity showed a 40% decrease over control in CHGP12 treated plants compared to the control in a glasshouse experiment. Moreover, CHGP12 also exhibited a significant increase in total biomass of the plants namely, root and shoot growth parameters, stomatal conductance, and photosynthesis rate. CONCLUSION: In conclusion, our findings suggest that B. thuringiensis CHGP12 is a promising strain with high antagonistic and growth-promoting potential against Fusarium wilt of chickpea. © 2022 Society of Chemical Industry.