First Report of Leaf Brown Spot Caused by Diaporthe phoenicicola on Pachira glabra in China.

Pachira glabra is an increasingly important ornamental landscape tree in southern China. In August 2022, brown spots were observed on P. glabra leaves in Xiangtan City, Hunan Province, China (27.932°N, 113.020°E), affecting up to 40% of the 792 trees surveyed. On each diseased tree, nearly 30% leaves had symptoms, with an average severity of 21.2 ± 5.8% (n=100). The disease initially started as small yellow lesions along leaf margins, which later progressed to pale brown to brown with dark brown borders, eventually coalescing into large necrotic areas. Thirty symptomatic leaf samples (2 × 2 mm) were surfaced-sterilized in 75% ethanol for 10 s, 2% NaOCl for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in dark. Eight morphologically similar isolates were obtained from diseased leaf samples through single-spore isolation. On PDA, colonies initially appeared white, turning gray, while the reverse developed a pale yellowish hue. Aerial mycelia were white, cottony, and developed visible black pycnidia with oil droplets at maturity. The α-conidia were unicellular, hyaline, aseptate, oval or fusiform, usually with 1 or 2 guttule(s) and rounded at each end. These conidia were 5.3-8.6 × 1.7-2.5 µm (avg. 6.7 × 2.2 µm, n = 100) and present more frequently than ß-conidia.The ß-conidia were unicellular, hyaline, aseptate, filiform, straight or hamate, eguttulate, 14.6-23.3 × 0.4-1.3 µm (avg. 18.4 × 0.9 µm, n = 30). Morphologically, the fungi were identified as Diaporthe sp. (Udayanga et al. 2014). For molecular identification, the internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), calmodulin (CAL), tubulin 2 (TUB2), and histone H3 (HIS3) sequences of all isolates were amplified from genomic DNA, using primers ITS4/ITS5 (White et al. 1990), TEF-2/728F and CALD-38F/CALD-752R (Carbone and Kohn 1999), Bt2a/Bt2b and H3-1a/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively. The GenBank accession numbers for a representative isolate gpg2023-1 were OR533573 (ITS), OR570887 (EF1-α), OR570888 (TUB2), OR570890 (CAL), and OR570889 (HIS3). BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences were 99%, 99%, 99%, 99%, and 98% identity, respectively, with those of Diaporthe phoenicicola (GenBank: KC343032.1, KC343758.1, KC344000.1, KC343516.1, and KC343274.1). To confirm the pathogen's identity, phylogenetic analysis using MEGA7.0 based on Maximum Likelihood was constructed. Isolate gpg2023-1 clustered with D. phoenicicola. Based on morphological and molecular data, the fungus was identified as D. phoenicicola. Next, pathogenicity tests were performed three times on one-year-old potted P. glabra plants. For each isolate, twelve healthy leaves on each of three plants were either wounded by a sterile needle or left unwounded, and then sprayed with a conidial suspension (1×106 conidia/ml) for each isolate. Control plants received with sterile water only. Plants were kept in a greenhouse at 25°C, 80% relative humidity, with a 12-h photoperiod. All wounded, inoculated leaves developed brown spot symptoms similar to those observed in the field with six days, while unwounded leaves and control plants remained symptom-free. The fungus was reisolated from all diseased leaves, fulfilling Koch's postulates and proving D. phoenicicola as the causative agent of this brown spot disease on P. glabra. While D. pachirae has been reported to cause leaf spot on P. glabra in Brazil (Milagres et al. 2018), this study marks the first report of D. phoenicicola causing leaf brown spot on P. glabra in China. This finding can help develop control strategies for this disease.

First Report of White Leaf Spot Caused by Hypomontagnella monticulosa on Pachira glabra in China.

Pachira glabra Pasq. is an ornamental tree widely distributed in tropical and subtropical regions of China. In August 2021, an unknown leaf spot was observed on P. glabra in Xiangtan County, Hunan, China (27.976°N, 113.041°E). Over 1,200 plants were evaluated, and up to 20% of the plants were diseased. In moderately diseased plants, approximately one third of leaves had symptoms with the disease severity estimated to be 31.6 ± 9.4% (n=100). The symptoms first appeared as pale yellow to yellow small dots often confined between leaf veins. These dots gradually enlarged, and coalesced into large pale or white spots with brown borders and yellow halo. In severe infections, early leaf death and defoliation occurred. Thirty lesions (2 × 2 mm) collected from ten trees were sterilized in 75% ethanol for 15 s, 5% sodium hypochlorite for 15 s, rinsed in sterile water three times, placed on oatmeal agar medium (OA) plate with lactic acid (3 ml/liter), and incubated at 28°C for 15 days. After incubation, five isolates with a similar morphology were obtained by single-spore culture. Colonies on OA were white and then turned pale grey. Pigments on the reverse side were pale brown. Conidiophores were hyaline, smooth to finely roughened, usually with virgariella-like branching patterns. Conidiogenouscells were hyaline, smooth, and measured 13.9 to 53.8 long and 1.5 to 2.3 µm wide (average 30.8 × 2.0, n=50). Conidia were single-celled, transparent, smooth, ellipsoid to obovoid, 2.3 to 4.6 × 1.7 to 3.1 µm (average 3.1 × 2.3, n=100) in measurement. For further molecular identification, internal transcribed spacer (ITS), ß-tubulin (TUB2), RNA polymerase II (RPB2), and large ribosomal subunit (LSU) genes of a representative isolate TT422 were amplified from genomic DNA, using primers ITS1/ITS4 (Mills et al. 1992), T1/T22 (O'Donnell et al. 1997), RPB2-5F/7cR (Liu et al. 2000), and LROR/LR7 (Rehner et al. 1994), respectively. Sequences of ITS (accession no. OM070368), TUB2 (OM201746), LSU (OM070369), and RPB2 (OM141478) from the isolate TT422 showed >98% identity where sequences overlapped to the reference strain of Hypomontagnella monticulosa MUCL 54604 (KY610404, KX271273, KY624305, and KY610487). Concatenated sequences were used for a phylogenetic analysis based on Maximum Likelihood using MEGA7. Based on morphological and molecular data, the isolate TT422 was identified as H. monticulosa (Ju & Rogers 1995; Lambert et al. 2019). Pathogenicity tests were performed three times on healthy leaves using the isolate TT422. Three leaves on one-year-old plants were slightly wounded by a sterile needle, and sprayed with conidial suspension (1×106 conidia/ml, containing 0.05% Tween 20) . Control leaves were sprayed with sterile water containing 0.05% Tween 20. All plants were kept in a greenhouse for 24 h at 28°C and 80% relative humidity, with a 16-h photoperiod and then transferred to natural conditions. All inoculated leaves developed white leaf spot symptoms after 7 days similar to those observed in the field, whereas no visible symptoms appeared on the control leaves. H. monticulosa strains were reisolated from all symptomatic leaves, fulfilling Koch's postulates. H. monticulosa isolated from marine or endophytic origin has been reported to produce bioactive metabolites with anticancer and microbial activities (Leman-Loubière et al. 2017; Lutfia et al. 2021; Zhang et al. 2021), but not as a phytopathogen. To our knowledge, this is the first report of H. monticulosa causing white leaf spot on P. glabra in China and worldwide.

First Report of Colletotrichum siamense Causing Leaf Anthracnose on Pachira glabra in China.

Pachira glabra Pasq.is an important landscape tree in southern China due to its ornamental value. Between March and April - 2021, anthracnose-like symptoms on P. glabra leaves were found in the botanical garden (27.904°N, 112.918°E) of Hunan University of Science and Technology located in Xiangtan of Hunan Province. Over 700 plants were evaluated, and up to 30% of the plants were symptomatic. On each plant, approximately 22% leaves had symptoms. Disease severity was estimated to be 15.6 ± 6.1% (n=100) in moderately diseased plants. Initially, subcircular or irregular shaped, water-soaked spots with pale green to yellow centers appeared mostly along leaf margins. Later, theses spots turned light brown to dark brown with black borders, gradually enlarged, and often coalesced into large sunken, necrotic areas, leading to early leaf death and abscission. Thirty lesions (2 × 2 mm) collected from ten trees were sterilized in 75% ethanol for 10 s, 2% sodium hypochlorite for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA) with lactic acid (3 ml/liter), and incubated at 28°C for 5 days. After incubation, six isolates with a similar morphology were obtained by single-sporing. Colonies on PDA were white and with age produced a light brown pigmentation on the underside of the colony. Acervuli present in aged cultures, brown to black, circular to subcircular and measured 31.9 to 108.7 µm (71.4 ± 6.2 µm, n=30). Conidia were single-celled, transparent, smooth, fusiform to cylindrical with obtuse to slightly ronded ends, and measured 7.8 to 11.1 µm long and 2.5 to 3.1 µm wide (9.3 ± 1.0 × 2.9 ± 0.7, n=100). For further molecular identification, Internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate (GAPDH), calmodulin (CAL), and beta-tubulin (TUB2) genes of the isolates were amplified from genomic DNA, using primers ITS1/ITS4 (Mills et al. 1992), GDF/GDR (Cannon et al. 2012), ACT-512F/ACT-783R, CL1CF/CL2CR (Weir et al. 2012), and T1F/T22R (O'Donnell et al. 1997), respectively. Sequences of ITS (accession no. OM074029), ACT (OM190777), GAPDH (OM190778), CAL (ON210110), and TUB2 (ON210109) from CS-1 showed >98% identity where sequences overlapped to the reference strain of Colletotrichum siamense CBS 130420 (JX010259.1, JX009549.1, JX009974.1, JX009713.1 and JX010415.1). Concatenated sequences were used for a phylogenetic analysis based on Maximum Likelihood using MEGA-X. Based on morphological and molecular data, isolate CS-1 was identified as C. siamense (Cannon et al. 2012). . Pathogenicity tests were performed three times on healthy leaves using isolate CS1. Ten leaves on one-year-old plants were either slightly wounded by a sterile needle or unwounded, and inoculated with 10 µl of conidial suspension (1×106 conidia/ml, containing 0.05% Tween 20) per wound. The control plants were treated with sterile water. All plants were kept in a greenhouse for 24 h at 28°C and 80% relative humidity, with a 12-h photoperiod and then transferred to natural conditions. All wounded, inoculated leaves developed leaf spot symptoms after 14 days similar to those observed in the field, whereas no visible symptoms appeared on the intact and noninoculated leaves. C. siamense strains were reisolated from all symptomatic leaves, fulfilling Koch's postulates. C. siamense has been reported as a causal agent of anthracnose associated with diverse species (Udayanga et al. 2013), but not including P. glabra. To our knowledge, this is the first report of C. siamense causing anthracnose on P. glabra.

Chemical constituents and gastro-protective potential of Pachira glabra leaves against ethanol-induced gastric ulcer in experimental rat model.

Gastric ulcer is a very common illness that adversely affects a significant number of people all over the globe. Phytochemical investigation of P. glabra leaf alcohol extract (PGLE) resulted in the isolation and Characterization of a new nature compound, quercetin-3- O-α -L-rhamnosyl-(1'''-6'')-(4''- O -acetyl)-ß -D-galactoside (4), in addition to seven known compounds. They are ferulic acid (1), p- coumaric acid (2), quercetin 3-O-α-L-rhamnoside-3'-O-ß-D-glucoside (3), quercetin-3- O-α -L-rhamnosyl-(1'''-6'')-(4''- O -acetyl)- ß -Dgalactoside (4), quercetin-3- O-ß -D-galactoside (5), 7-hydroxy maltol-3-O-ß-D-glucoside (6), maltol-3- O-ß -D-glucoside (7), and methyl coumarate (8) that were first to be isolated from the genus Pachira. PGLE demonstrated in vitro anti-Helicobacter pylori activity. Moreover, the in vivo gastroprotective assessment of PGLE at different dosses, 100, 200, and 400 mg/kg against ethanol induced ulceration revealed a dose-dependent gastroprotection comparable to omeprazole. PGLE attenuated gastric lesions and histopathological changes triggered by ethanol. Interestingly, PGLE exhibited an anti-inflammatory effect through down-regulating the expression of nuclear factor-ĸB and pro-inflammatory enzyme cyclooxygenase-2 in the ulcer group. It also hindered apoptosis through decreasing Bax and increasing Bcl-2 expression hence decreasing Bax/Bcl2 ratio with a subsequent reduction in caspase 3 expression. Collectively, P. glabra is a rich reservoir of various phytochemicals reflecting a promising potential for alleviation of gastric ulcer through the mediation of inflammatory and apoptotic cascades.

Effects of extraction parameters on the yield of oils from non-edible seeds of Bauhinia variegata and Pachiraglabra.

The influence of extraction temperature, seed age, and extraction time and their interactions on the Bauhinia variegata seed oil (BVSO) and Pachira glabra seed oil (PGSO) yield was studied using the response surface methodology (RSM). The BVSO and PGSO obtained were characterized to determine their suitability for use as biofuel. Numerically predicted optimum values for the extraction process using the RSM model proved to be a one-week-old seeds, a 10 h extraction time and a 60 °C temperature with a 47.05 % PGSO yield, and a one-week-old seed, a 6.5 h extraction period and 60 °C temperature, with a 23.1 % BVSO yield. Performance evaluation of the models by coefficient of determination (R2), Adjusted R2, and absolute average deviation (AAD) showed that the RSM model (R2 = 0.99, Adjusted R2 = 0.99, AAD = 0.07 % for BVSO yield, and R2 = 0.99, Adjusted R2 = 0.99, AAD = 0.01 % for PGSO yield) was satisfactory, reliable, and flexible. The physicochemical properties of BVSO and PGSO, i.e. acidity index (4.63 mg KOH/g and 27.21 mg KOH/g) and kinematic viscosity (3.24 mm2/s and 12.45 mm2/s), reveal the need for post-treatment of oils for use as additives to conventional fuels. Other physicochemical properties obtained, such as oxidative stability, higher heating value, cetane number, flash point, iodine value, and saponification value, demonstrate that these oils are excellent potential sources for biodiesel production.

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra.

Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.

Calidion bombacis, a new combination for Uredo bombacis with the record of Bombacopsis glabra (Bombacaceae) as a new host from Brazil.

The new combination Calidion bombacis is proposed for the rust species formerly known as Uredo bombacis. This nomenclatural change is based on the examination of newly collected material of this fungus from a new host, Bombacopsis glabra from Brazil, and reexamination of the isotype. Until now this fungus was only known to occur in Asia (China, India and Sri Lanka). Therefore, this is also the first record of this fungus from the Neotropics.

Calidion bombacis, a new combination for Uredo bombacis with the record of Bombacopsisglabra (Bombacaceae) as a new host from Brazil / Calidion bombacis, uma nova combinação para Uredo bombacis e o relato de Bombacopsisglabra (Bombacaceae), como um novo hospedeiro no Brasil

The new combination Calidion bombacis is proposed for the rust species formerly known as Uredobombacis. This nomenclatural change is based on the examination of newly collected material of this fungus from a new host, Bombacopsisglabra from Brazil, and reexamination of the isotype. Until now this fungus was only known to occur in Asia (China, India and Sri Lanka). Therefore, this is also the first record of this fungus from the Neotropics.

A combinação nova Calidion bombacis é proposta para a ferrugem anteriormente conhecida como Uredo bombacis. Esta modificação nomenclatural é proposta baseada no exame de material deste fungo coletado em um novo hospedeiro, Bombacopsisglabra (Bombacaceae) no Brasil e reexame do isotipo. Até então este fungo tinha sido relatado apenas na Ásia (China, Índia e Sri Lanka). Portanto, este é o primeiro relato deste fungo nos Neotrópicos.