First Report of Diaporthe eres Causing Leaf Spot on Pseudocydonia sinensis in China.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and ß, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 µm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 µm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The ß-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

First Report of Botryosphaeria dothidea Causing Leaf Blight on Cornus officinalis in Jiangsu Province, China.

Cornus officinalis Sieb. et Zucc., belonging to the family Cornaceae, is often used as an ornamental plant and is widely distributed in Shandong, Jiangsu, and Zhejiang provinces and other places in China. Since 2020, a new disease with high incidence has been found in Xuanwu Lake Park (32°04'34.53″N 118°48'42.06″E) in Nanjing, Jiangsu Province, China. The symptoms began as small brown lesions formed along the leaf tips, which gradually expanded and became dark brown with a light brown border. A survey of C. officinalis trees in Xuanwu Lake Park showed that approximately 90% of thirty trees were infected, which decreased the ornamental value of C. officinalis. Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated in 90% of the tissues. Pure cultures were obtained by monosporic isolation. A representative isolate, SZY 2-2, was used for morphological and molecular characterization. The colonies were initially white, gradually turning gray green to black with copious gray aerial mycelium after 1 week in culture. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.6±1.9×7.2±0.56 µm (n = 50). The morphology of SZY 2-2 matched the description of Botryosphaeria dothidea (Slippers et al. 2004). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-a) gene, and beta-tubulin gene (TUB), were amplified from isolate SZY 2-2 with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Koho 1999), and ßt2a/ßt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (ON171471 for ITS, ON185540 for EF1-a, and ON185541 for TUB). A BLAST search of GenBank showed that ITS, EF1-a and TUB sequences of SZY 2-2 were similar to those of B. dothidea MN633360 (identity=517/517 bp; 100%), MK783294 (identity=299/299 bp; 100%), and KF005081 (identity=461/461 bp; 100%), respectively. The morphological and molecular results identified the isolate as B. dothidea (Zhai et al. 2014). To fulfill Koch's postulates, a pathogenicity test was conducted using three C. officinalis plants. Five leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of B. dothidea from a 5-day-old culture grown on PDA, and inoculation with sterile PDA plugs on different leaves of the same tree served as negative controls. The leaves were enclosed in plastic bag along with the branches with a wet cotton ball inside. Sterile H2O2 was sprayed into the plastic bags to keep moisture conditions.Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and had morphology as characterized before, thus fulfilling Koch's postulates. B. dothidea is known as a ubiquitous fungus and operates as both an endophyte and an opportunistic pathogen of trees (Slippers and Wingfield 2007, Zhao et al 2020). Stress factors that predispose trees to disease expression by B. dothidea include drought, defoliation (Theodore et al. 1997), competition, and physical damage (Slippers and Wingfield 2007). This is consistent with the occurrence of the disease in September and association of B. dothidea with the presence of wounds. More investigation is needed to determine the relationship between possible endophytic growth of B. dothidea on C. officinalis and the leaf blight found in Jiangsu Province.

First Report of leaf spot disease caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China.

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 µL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.

[Isolation, identification, and degrading effect of a pyrene-degrading strain SE12].

Using pyrene as the sole carbon and energy source, and by the method of plate sublimation, a strain SE12 was isolated from a contaminated soil near Woniushan Coking Plant in Xuzhou, China. According to the morphological, biochemical, and 16S rDNA analyses, this strain was identified as Mycobacterium sp., with 98% of homology to the rapid-growth nonpathogenic strain M. austroafricanum ATCC 33464. The optimum pH and temperature for the degradation of pyrene by SE12 were pH 9 and 30 degrees C. When the soil samples were added with 100 and 200 mg x kg(-1) of pyrene and inoculated with 10(7) CFU x g(-1) of SE12, the degradation rates of pyrene reached to 97% and 99%, respectively after 28 days incubation at 30 degrees C. By using primer-pairs nidAF/nidAR and nidBF/nidBR for amplification of ring-hydroxylating dioxygenase genes, it was shown that SE12 had the fragments of encoded large and small subunits of dioxygenase genes. Sequence analysis showed that these fragments were highly homologous to the known dioxygenase genes from pyrene-degrading Mycobacteria sp.

[Turbidity removal of summer high algae content Taihu Lake raw water using composite coagulants of polyaluminum chloride and polydimethyldiallylammonium chloride].

A series of stable composite coagulants was prepared using polyaluminum chloride (PAC) and polydimethyldiallylammonium chloride (PDM) with different intrinsic viscosity as raw materials. The enhanced coagulation processes of summer high algae content Taihu lake raw water by using the composite coagulants were reported in this paper. Jar tests were used to study the influences of the composite ratios of PAC to PDM and the intrinsic viscosity of PDM on the turbidity removal and precipitating performance. The results showed that for the summer Taihu raw water with temperature of 28-30 degrees C and turbidity of 30-33 NTU, algae content of 2.6 x 10(7) cells/L. When 2NTU turbidity of supernatant from settlement tank was required according to the current process of potable water plant in Wuxi, the dosage of 7.00 mg/L PAC was necessary, the PAC/PDM composite coagulants with the ratios (m/m) of 20:1, 10:1, 5:1 only needed dosage ranges of 3.00-2.83 mg/L, 3.50-3.49 mg/L, 5.37-4.67 mg/L with the increasing of PDM intrinsic viscosity from 0.52 dL/g to 2.46 dL/g. And compared with PAC only used, composite coagulants could decrease the PAC dose by 57.14%-59.57%, 50%-50.14%, 23.29%-33.29% correspondingly. When residual turbidity of 1 NTU was required, the dosage of 10 mg/L were necessary for the treatment with PAC , composite coagulants with the ratios(m/m) of PAC to PDM of 20:1, 10:1, 5:1 and the different intrinsic viscocity of PDM needed dosage of 8.33-3.91 mg/L, and could decrease the PAC dose by 16.7%-60.9%. Therefore, the efficiency turbidity removal and precipitating performance of composite coagulants were remarkably, and the lower the ratios of PAC to PDM and the higher the intrinsic viscosity of PDM, the higher efficiency of turbidity removal.