Assessment of bioremediation potential of Calotropis procera and Nerium oleander for sustainable management of vehicular released metals in roadside soils.

Land transportation is a major source of heavy metal contamination along the roadside, posing significant risks to human health through inhalation, oral ingestion, and dermal contact. Therefore, this study has been designed to determine the concentrations of vehicular released heavy metals (Cd, Pb, Ni, and Cu) in roadside soil and leaves of two commonly growing native plant species (Calotropis procera and Nerium oleander).Two busy roads i.e., Lahore-Okara road (N-5) and Okara-Faisalabad roads (OFR) in Punjab, Pakistan, were selected for the study. The data were collected from five sites along each road during four seasons. Control samples were collected ~ 50 m away from road. The metal content i.e. lead (Pb), cadmium (Cd) nickel (Ni) and copper (Cu) were determined in the plant leaves and soil by using Atomic Absorption Spectrophotometer (AAS). Significantly high amount of all studied heavy metals were observed in soil and plant leaves along both roads in contrast to control ones. The mean concentration of metals in soil ranged as Cd (2.20-6.83 mg/kg), Pb (4.53-15.29 mg/kg), Ni (29.78-101.26 mg/kg), and Cu (61.68-138.46 mg/kg) and in plant leaves Cd (0.093-0.53 mg/kg), Pb (4.31-16.34 mg/kg), Ni (4.13-16.34 mg/kg) and Cu (2.98-32.74 mg/kg). Among roads, higher metal contamination was noted along N-5 road. Significant temporal variations were also noted in metal contamination along both roads. The order of metal contamination in soil and plant leaves in different seasons was summer > autumn > spring > winter. Furthermore, the metal accumulation potential of Calotropis procera was higher than that of Nerium oleander. Therefore, for sustainable management of metal contamination, the plantation of Calotropis procera is recommended along roadsides.

First Report of Fruit Rot Caused by Diaporthe amygdali on Papaya in Punjab, Pakistan.

Papaya (Carica papaya L.) is grown widely in tropical and sub-tropical regions (Ahmed et al. 2008). In Pakistan, papaya production and consumption are increasing due to its medicinal, nutritional, pharmacological properties and a rich source of antioxidant, vitamin B, potassium, and magnesium. In November 2021, 26 to 35% incidence of fruit rot was observed in 15 fields of Lahore, a district of Punjab, Pakistan. Affected fruit developed circular, gray-to-brown lesions (8 to 10 mm in diameter) with white mycelia forming on the surface of lesions. In advanced stages of the disease, the lesions enlarged in size and led to the rot of entire fruit. To isolate the causal agent, small tissue segments (1 to 2 cm) were excised from 15 symptomatic fruit, surface disinfected with 1% NaClO for 30 s, rinsed with sterile distilled water three times, air dried in laminar flow hood, aseptically transferred onto petri dishes containing potato dextrose agar (PDA) and incubated at 25℃ for 5 days with a 12-h photoperiod. Eleven isolates were obtained that produced white mycelia on PDA. Flask-shaped, dark-pigmented pycnidia formed on PDA after 18 days of incubation at 25°C, which produced α-conidia measuring 4.1 to 7.2 × 1.5 to 3.0 µm and ß-conidia measuring 16.4 to 25.5 × 1.0 to 1.6 µm (n = 40). α-conidia were hyaline, fusiform, and single-celled, whereas ß-conidia were one-celled, hyaline, and filiform. The morphological characteristics of the fungus were compatible with a Diaporthe species (Gomes et al. 2013). The internal transcribed spacer region (ITS) (OM865414 and OM865415), translation elongation factor 1-alpha (tef1) (OM831226 and OM831229), and histone H3 (HIS) (OM831227 and OM831228) of two representative isolates (UO02 and UO03) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and CYLH3F/H3-1b (Chaisiri et al. 2021), respectively. Blast searches showed 99 to 100% nucleotide identity with reference sequences of several Diaporthe amygdali deposited in NCBI GenBank, including the ex-type strain CBS 126679. A pathogenicity test was also performed on harvested fruit of papaya cv. Bombay using isolates UO02 and UO03. Ten mature and healthy papaya fruit were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water and dried. Each fruit was wounded twice with a sterile scalpel (4 to 5 mm incision on the peel) and a 5-mm agar disc with mycelia of each isolate was separately placed in each wound. The wounds were wrapped with Parafilm following inoculation. Sterile PDA plugs were used in separate inoculated controls. All wounds were sealed with parafilm. All fruit were maintained in plastic boxes at 25°C with 80% relative humidity. After 6 days of incubation, rot symptoms similar to those appearing on naturally-infected fruit were observed on inoculated fruits while controls remained asymptomatic. The experiment was repeated twice with similar findings. Diaporthe amygdali was re-isolated (100%) from inoculated fruit and the pathogen identification was confirmed by morphological and molecular analysis, thus fulfilling Koch's postulates. Previously, the pathogen has been reported as a causal agent of canker and shoot blight disease in other countries (Ko and Sun, 2003; Beluzan et al. 2021). To our knowledge, this is the first report of D. amygdali on papaya in Punjab Province of Pakistan. Papaya is an emerging fruit crop in Punjab Province and it is important to further investigate the presence of this pathogen in other papaya orchards of the province since D. amygdali may cause rapid disease outbreaks resulting in severe losses.

Triacontanol regulates morphological traits and enzymatic activities of salinity affected hot pepper plants.

Potential role of triacontanol applied as a foliar treatment to ameliorate the adverse effects of salinity on hot pepper plants was evaluated. In this pot experiment, hot pepper plants under 75 mM NaCl stress environment were subjected to foliar application of 25, 50, and 75 µM triacontanol treatments; whereas, untreated plants were taken as control. Salt stress had a significant impact on morphological characteristics, photosynthetic pigments, gas exchange attributes, MDA content, antioxidants activities, electrolytes leakage, vitamin C, soluble protein, and proline contents. All triacontanol treatments significantly mitigated the adversative effects of salinity on hot pepper plants; however, foliar application triacontanol at 75 µM had considerably improved the growth of hot pepper plants in terms of plant height, shoot length, leaf area, plant fresh/dry biomasses by modulating above mentioned physio-biochemical traits. While, improvement in gas exchange properties, chlorophyll, carotenoid contents, increased proline contents coupled with higher SOD and CAT activities were observed in response to 75 µM triacontanol followed by 50 µM triacontanol treatment. MDA and H2O2 contents were decreased significantly in hot pepper plants sprayed with 75 µM triacontanol followed by 50 µM triacontanol foliar treatment. Meanwhile, root and shoot lengths were maximum in 50 µM triacontanol sprayed hot pepper plants along with enhanced APX activity on exposure to salt stress. In crux, exogenous application triacontanol treatments improved hot pepper performance under salinity, however,75 µM triacontanol treatment evidently was more effective in mitigating the lethal impact of saline stress via controlling the ROS generation and increment in antioxidant enzyme activities.

Triacontanol modulates salt stress tolerance in cucumber by altering the physiological and biochemical status of plant cells.

Cucumber is an important vegetable but highly sensitive to salt stress. The present study was designed to investigate the comparative performance of cucumber genotypes under salt stress (50 mmol L-1) and stress alleviation through an optimized level of triacontanol @ 0.8 mg L-1. Four cucumber genotypes were subjected to foliar application of triacontanol under stress. Different physiological, biochemical, water relations and ionic traits were observed to determine the role of triacontanol in salt stress alleviation. Triacontanol ameliorated the lethal impact of salt stress in all genotypes, but Green long and Marketmore were more responsive than Summer green and 20252 in almost all the attributes that define the genetic potential of genotypes. Triacontanol performs as a good scavenger of ROS by accelerating the activity of antioxidant enzymes (SOD, POD, CAT) and compatible solutes (proline, glycinebetaine, phenolic contents), which lead to improved gas exchange attributes and water relations and in that way enhance the calcium and potassium contents or decline the sodium and chloride contents in cucumber leaves. Furthermore, triacontanol feeding also shows the answer to yield traits of cucumber. It was concluded from the results that the salinity tolerance efficacy of triacontanol is valid in enhancing the productivity of cucumber plants under salt stress. Triacontanol was more pronounced in green long and marketer green than in summer green and 20252. Hence, the findings of this study pave the way towards the usage of triacontanol @ 0.8 mg L-1, and green long and marketer genotypes may be recommended for saline soil.

First Report of Lasiodiplodia pseudotheobromae Causing Stem End Rot of Mango Fruit in Pakistan.

Mango (Mangifera indica L.) is considered a desirable fruit in international markets and is grown throughout tropical and sub-tropical countries around the world (Alemu, 2014). Stem end rot is the most damaging and complex postharvest disease of mango, resulting in losses of up to 40% in Pakistan, which is the leading producer and exporter (Alam et al. 2017). A field survey was conducted in June of 2017 and 2018 in the Rahim Yar Khan and Multan- major mango producing regions of Punjab Province. After mature but unripe mango fruit (cv. Samar Bahisht Chaunsa) were stored at 12°C for 2 weeks to permit ripening, water-soaked, dark brown to purplish black decay began to appear around the stem end portion. The decay gradually enlarged and covered the whole fruit after 7 days. Disease incidence was estimated at 30%. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected with 1% sodium hypochlorite for 2 min, rinsed three times in sterilized distilled water, air dried, and then placed aseptically onto potato dextrose agar (PDA) medium and incubated at 25°C under a 12-h light/dark photoperiod for 7 days. Twelve single-spore isolates with similar morphology were isolated from the infected tissues. Initially the fungus produced thick, fluffy and greyish-white aerial mycelium, that later turned into dark gray colonies. Conidia were unicellular, ellipsoidal, and initially hyaline, but with age became dark brown and developed a central septum. Conidia measured 24.5 to 31.5 × 11.4 to 15.7 µm (n = 60). Conidiophores were inflated at their base with one diaphragm which reduced to conidiogenous cells. Conidiogenous cells were hyaline and cylindrical. On the basis of morphological characteristics, the fungus was tentatively identified as Lasiodiplodia sp., a member of the family Botryosphaeriaceae (Alves et al. 2008). For molecular identification, genomic DNA was extracted from mycelium following the CTAB method. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn searches of sequences revealed 99% to 100% identity with the reference sequences of various Lasiodiplodia pseudotheobromae isolates (GenBank accession nos. MH057189 for ITS; MN638768 for TEF-1a). The sequences were deposited in GenBank (accession nos. MW439318, MW433883 for ITS; and MW463346, MW463347 for TEF-1a). To fulfill Koch's postulates, a suspension of 105 conidia/ml from a 7-day-old culture of L. pseudotheobromae was used to inoculate fully mature but unripe mango fruit (cv. Samar Bahisht Chaunsa). Fruit were pricked with a sterilized needle to a depth of 4 mm at the stem end portion, injected with 50 µl of the prepared spore suspension (Awa et al. 2012), and stored at 12°C for 3 weeks under 70 to 80% RH. Twenty mango fruit were inoculated, and 10 were inoculated with sterile water only. After 15 days, most fruit showed typical symptoms at the stem end. Reisolations from symptomatic fruit following the procedures described above for isolating and identifying the fungal cultures from infected field samples, consistently yielded a fungus identical to L. pseudotheobromae. Control fruit remained disease-free. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), this is the first report of the pathogen causing stem end rot disease of mango in Pakistan. This report is important for the new studies aiming at management of stem end rot disease of mango caused by L. pseudotheobromae in Pakistan.

First Record of Chaetomium globosum Causing Leaf Spot of Pomegranate in Pakistan.

Pomegranate (Punica granatum L.) is a non-climacteric and a favorite fruit of tropical, sub-tropical and arid regions of the world. During a survey in autumn 2019, leaf lesions were observed on plants (cv. Kandhari) in different orchards of Muzaffargarh (30°4'27.7572″ N, 71°11'4.7544″ E), a major pomegranate-producing region in Punjab Province. Disease incidence ranged from 17 to 20%. Leaf lesions were initially small (1 to 3 mm in diameter), round, purple or reddish-brown, scattered spots. At later stages, spots increased in size and the centers of mature lesions became dark red or black with fungal sporulation. To isolate the pathogen, samples of leaf (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface disinfected in 75% alcohol for 30 s, sterilized with 6% sodium hypochlorite for 3 min, washed with sterile distilled water three times, air dried in laminar flow hood, and cultured on potato dextrose agar (PDA). After one week of incubation at 25 ± 2°C with a 12-h photoperiod, fungal colonies developed, which were initially white and became pale yellow with olivaceous green mycelium after 20 days. On PDA, ascomata were olivaceous green, with a papillate ostiole, globose or ovoidal to obovoidal (155 to 220 × 120 to 240 µm, n=50). Terminal and lateral setae were abundant, brown, and tapering toward the tips (4 to 6 µm, n=50). Asci were greenish and lemon-shaped (6 to 8 × 9 to 13.5 µm, n=50). Ascospores were limoniform and olivaceous gray-brown (10 to 11.5 × 7 to 9 µm, n=50). These morphological characteristics were consistent with the morphology of Chaetomium globosum (Lan et al. 2011; Wang et al. 2016). Genomic DNA was extracted from two isolates and identification of the pathogen was confirmed by amplification and sequencing of the internal transcribed spacer region (ITS) and the partial translation elongation factor 1-α (TEF1) gene using ITS1/ITS4 (White et al. 1999) and EF1-983F/EF1-2218R primers (Wang et al. 2016), respectively. The sequences of the PCR products were deposited in GenBank with accession numbers MW522514, MW522352 (ITS), and MW530423, MW530424 (TEF1). BLAST results of the obtained sequences of the ITS and TEF1 genes revealed 100% (513/513 bp) and 99.78% (927/929 bp) similarity with those of C. globosum in GenBank (ITS: KX834823 and KT898637, and TEF1: MG812564 and KC485028). To confirm pathogenicity, inoculum was prepared by harvesting conidia from 10-day-old culture grown in PDA. The surface-disinfected (70% ethyl alcohol, 30 s) leaves of ten 1-year-old seedlings (cv. Kandhari) were sprayed with a spore suspension (1×106 conidia/ml). Leaves of ten seedlings sprayed with sterile distilled water served as controls. All seedlings were covered with plastic bags and placed in a greenhouse at 26°C with 12 h photoperiod. After eight days, symptoms on inoculated leaves were similar to those observed in the orchards; no symptoms were observed on controls. The fungus was reisolated from all symptomatic tissues. C. globosum has been reported on Punica granatum (Guo et al. 2015), Cannabis sativa (Chaffin et al. 2020) and Brassica oleracea (Zhu et al. 2020). This is the first report of C. globosum causing leaf spot on pomegranate in Pakistan. This finding suggests a potential threat to pomegranate production in Pakistan and further studies should focus on effective prevention and control practices of this disease.

First Record of Colletotrichum gloeosporioides Causing Anthracnose of Banana in Pakistan.

Banana (Musa spp.) is one of the most widely grown and consumed fruits in Pakistan and all around the world due to their distinct aroma and taste. In 2018, anthracnose symptoms were observed on banana fruit harvested from different plantations of Sindh- a major banana producing Province of Pakistan. Approximately, 25% of banana fruit collected from different plantations were infected. The symptoms consisted of small brown to reddish-brown spots on the fruit surface and then became sunken lesions as the disease progressed. To identify the pathogen, infected tissues (5 mm in diameter) from the margin of the lesions were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 2 min, 70% ethanol for 30 s, and then rinsed twice with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 27°C for 5 days with 12 h light and darkness cycle. Colonies with a similar pattern were consistently isolated and all colonies were sub-cultured using the single-spore method. Colonies first appeared with white colored mycelium and later turned to dark gray. Conidia produced in acervuli were cylindric, hyaline, straight, and aseptate, with both ends rounded. Conidia measured 14.0 ± 0.5 × 3.4 ± 0.6 µm. Conidiomata were dark brown and spherical. On the basis of morphological characterization, the pathogen was identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Weir et al. 2012). Two independent isolates (PDL2031 and PDL2032) were used for further genetic analysis. The internal transcribed spacer (ITS) region and chitin synthase 1 (CHS-1) gene were amplified from genomic DNA using primer pairs of ITS1/ITS4 and CHS-79F/CHS-345R, respectively (White et al. 1990; Damm et al. 2012). The GenBank accession numbers (MW493198, MW504711 for ITS and MW530421, MW530422 for CHS-1) of the sequences exhibited 99% to 100% identity to multiple sequences of C. gloeosporioides. To conduct a pathogenicity test, 10 healthy fruits were selected and surface sterilized with 70% ethanol followed by a wash of sterilized water. The fruits were stabbed with a sterile needle and a drop of 20 µl of spore suspension (106 spores/ml) was placed on each wound independently. Meanwhile 10 fruits inoculated with sterile water were treated as controls. The fruits were incubated at 27°C with 90% relative humidity for 10 days. Inoculated fruits exhibited symptoms similar to the original infection. No visible lesions appeared on control fruit. C. gloeosporioides was successfully reisolated from the inoculated fruit, confirming Koch's postulates. Anthracnose of banana is known to be caused by C. musae, C. gloeosporioides, C. siamense, C. tropicale, C. chrysophilum, C. theobromicola, and C. scovillei (Kumar et al. 2017; Peres et al. 2001; Vieira et al. 2017; Zakaria et al. 2009; Zhou et al. 2017). To our knowledge, this is first report of anthracnose of banana caused by C. gloeosporioides in Pakistan. The new disease primarily reduces the quality and yield of Banana. Effective measures should be taken to manage this disease.