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 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 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 Report of Postharvest Stem End Rot of mango fruit (Mangifera indica) caused by Lasiodiplodia theobromae in China.

China is the second largest producer of mango in the world, a fruit has high nutritive value and a rich source of fiber (Kuhn et al., 2017). In late June 2019, a postharvest stem-end rot disease was observed in different local fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district of Beijing, China. Black rot symptomatic lesions were observed on the fruit surface which initially started from the stem end of the mango fruit (Fig. 1). Approximately 45 % of mango fruits were affected with the disease. Symptomatic portions from collected fruit samples (n=40) were cut into small pieces (2mm2), rinsed with 1% NaClO for 20s and then washed three times with sterilized distilled water (SDW) for surface disinfection. The disinfected pieces were then placed on sterilized filter paper for drying. Later, these pieces were placed on Potato Dextrose Agar (PDA) plates and incubated at 28°C for seven days. The resulting fungal colonies were purified by the single spore isolation technique. The isolated fungal colonies were initially greenish to gray in color, later turning olive-black to black. Conidia were dark brown in color, oval-shaped, two-celled and measured 22.4 to 25.7 (24.06 ± 0.15) µm in length and 10.2 to 12.8 (11.3 ± 0.13) µm in width (n=36). Based on the symptoms, culture morphology and microscopic characters, Lasiodiplodia theobromae was suspected as the causal agent, and similar results were reported by Pavlic et al., 2004 and Burgess et al., 2006. For molecular identification, a multi-locus sequence analysis approach was used. The Internal Transcribed Spacers (ITS) region, elongation factor 1 alpha (EF1-α) and ß-tubulin genes were amplified and sequenced using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), and Bt2a/Bt2b (Glass and Donaldson, 1995) primers respectively. The sequences of isolate MFT9 were deposited to GenBank (MW115977 (ITS), (MW118595 (EF1-α) and MW118596 (ß-tubulin). All sequences showed more than 99.5% similarity with reported sequences of Lasiodiplodia theobromae isolate IBL340 with accessions numbers KT247466 (ITS), KT247472 (EF1-α) and KT247475 (ß-tubulin). Phylogenetic reconstruction based on Maximum Likelihood, using Mega X (Kumar et al., 2018), grouped isolate MFT9 with isolates representing L. theobromae. Pathogenicity testing was performed on 18 fresh, healthy, medium-sized mango fruits for each treatment to fulfill Koch's postulate. The fruits were disinfested with 1% NaClO and punctured with a sterilized needle to create approximately 2mm2 wounds for inoculation. Fruits were inoculated with 15µL of fresh inoculum (107 spores/mL) from isolate MFT9. Control fruits were inoculated with 15µL of SDW and both the inoculated and control fruits were incubated at 28°C for seven days of post inoculation. The rot lesions appeared at the point of inoculation and gradually spread on the fruit surface. The symptoms were similar to the symptoms observed on the original fruit samples (Fig. 2). This experiment was conducted three times under the same conditions, with control fruits remaining asymptomatic each time. The re-isolated fungus was identified as L. theobromae based on symptoms and morpho-molecular analysis, described above. L. theobromae is also reported as a causal agent responsible for a postharvest stem-end rot on Coconut in China (Zhang, et al., 2019). To our knowledge, this is the first report of L. theobromae causing postharvest stem-end rot of mango fruit in China. This finding suggests that L. theobromae is a potential problem for mango fruit production in China.

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 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.