Transcriptional dynamics reveals the asymmetrical events underlying graft union formation in pecan (Carya illinoinensis).

Grafting is a widely used technique for pecan propagation; however, the background molecular events underlying grafting are still poorly understood. In our study, the graft partners during pecan [Carya illinoinensis (Wangenh.) K. Koch] graft union formation were separately sampled for RNA-seq, and the transcriptional dynamics were described via weighted gene co-expression network analysis. To reveal the main events underlying grafting, the correlations between modules and grafting traits were analyzed. Functional annotation showed that during the entire graft process, signal transduction was activated in the scion, while messenger RNA splicing was induced in the rootstock. At 2 days after grafting, the main processes occurring in the scion were associated with protein synthesis and processing, while the primary processes occurring in the rootstock were energy release-related. During the period of 7-14 days after grafting, defense response was a critical process taking place in the scion; however, the main process functioning in the rootstock was photosynthesis. From 22 to 32 days after grafting, the principal processes taking place in the scion were jasmonic acid biosynthesis and defense response, whereas the highly activated processes associated with the rootstock were auxin biosynthesis and plant-type secondary cell wall biogenesis. To further prove that the graft partners responded asymmetrically to stress, hydrogen peroxide contents as well as peroxidase and ß-1,3-glucanase activities were detected, and the results showed that their levels were increased in the scion not the rootstock at certain time points after grafting. Our study reveals that the scion and rootstock might respond asymmetrically to grafting in pecan, and the scion was likely associated with stress response, while the rootstock was probably involved in energy supply and xylem bridge differentiation during graft union formation.

First Report of Alternaria Leaf Spot Caused by Alternaria alternata on Ulmus parvifolia in Jiangsu, China.

Ulmus parvifolia Jacq. is an important tree with ornamental value, which is widely planted in Hebei and southern regions of China. In September 2022, a leaf spot symptom was observed on about approximately 20% U. parvifolia seedlings growing a tree farm (20000 m2) of Jiangsu Academy of Forestry (118°45'57.30″E, 31°51'27. 94″N). Gray to black spots appeared on leaves of seedlings. Five diseased leaves were collected from five different seedlings. The pieces were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the darkness. Pure cultures were obtained by monosporic isolation. Six isolates with identical morphological features and the internal transcribed spacer (ITS) sequences were obtained (the isolate rate of 67%), and identified as Alternaria sp. A representative isolate, LY-1-1 was used for the further study. The colony of LY-1-1, growing on PDA was cotton-like and brown in color with gray-white aerial hyphae on their surfaces, and its reverse was dark grey. The conidia were ovate to pear-shaped, brown in color, with 1 to 4 transverse septa and 0 to 1 longitudinal septa, parietal cells extending into the beak, and measured 7.1 to 12.5×3.8 to 7.1 µm (n=35). These characteristics were consistent with the description of Alternaria sp. (Simmons 2007). The regions of ITS, large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), anonymous region OPA10-2 genomic sequence (OPA10-2), Alternaria 1 major allergen (Alta1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) genes (GenBank Accession No. OR047916, OR051904, OR047919, OR061065, OR061063, OR061064, and OR061062, respectively) were amplified (White et al. 1990; Woudenberg et al. 2015) and sequenced. These obtained sequences showed 99.86-100% similarity to the ITS (514/515 bp) of A. alternata isolate SPM-2 (OR378581), LSU (801/801 bp) of isolate B9 (OR366492), SSU (1019/1020 bp) of strain LSU0766 (MT000349), OPA10-2 (632/633 bp) of strain 19-1 (MN185000), Alta1 (470/470 bp) of strain CMML21-73 (OQ831518), GAPDH (177/177 bp) of isolate CS36-3 (KY814638), and TEF1 (240/240 bp) of isolate SY-6 (OP980553). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate LY-1-1 clustered in the A. alternata clade with 98% bootstrap support. Three 3-month-old U. parvifolia seedlings were wounded with a sterile needle and inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation on the right side with 20 µL of sterile water was treated as a control. All inoculated plants were incubated in a greenhouse at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical gray to black spots were found on the left sides of all inoculated leaves, and the control did not have any leaf spot symptoms. Subsequently, the same fungus was reisolated and identified based on morphological and molecular traits, fulfilling Koch's postulates. The A. alternata has been reported to cause leaf spot on pecan (Wu et al. 2020), fruit spot on olive (Alam et al. 2019) and fruit rot on lychee (Alam et al. 2017). However, there are no other reports of A. alternata on U. parvifolia in the world. Thus, this study provides an important reference for the biology, epidemiology of A. alternata.

The Bursaphelenchus xylophilus candidate effector BxLip-3 targets the class I chitinases to suppress immunity in pine.

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant-parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip-3. It was transcriptionally up-regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip-3 suppressed cell death triggered by the pathogen-associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase-3 domain is essential for immunosuppression. Silencing of the BxLip-3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis-related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two-hybrid and co-immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1-3 and PtChia1-4, as targets of BxLip-3. The expression of these two chitinases was up-regulated during B. xylophilus inoculation and inhibited by BxLip-3. Overall, this study illustrated that BxLip-3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine.

First report of Epicoccum latusicollum causing leaf spot on Lophatherum gracile in Jiangsu Province of China.

Lophatherum gracile Brongn. is an important Chinese herbal medicine. Since 2016, a leaf spot disease has appeared on L. gracile seedlings in the traditional Chinese medicine resource garden of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province (32.06°N, 118.83°E). About approximately 80% of the seedlings suffered from the disease. The disease spot usually starts from the leaf margin, round or irregular, with yellow halo at the edge of the lesion. To isolate the pathogen, four diseased leaves were collected from four different seedlings and there are 6 sections from each diseased leaf. The leaf sections were surface sterilized in 75% alcohol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA). Pure cultures were obtained by monosporic isolation. Eleven isolates were obtained (isolate rate of 55%) and identified as Epicoccum sp.. Thus, a representative isolate, DZY3-3 was used for the further study. After 7 days of culture, the colony produced white aerial hyphae, and reddish orange pigment on the underside. The chlamydospores were produced, either multicellular or unicellular. The colony produced pycnidia and conidia after nearly three weeks of cultivation on oatmeal ager OA. Conidia were unicellular, hyaline, oval, and were 4.9 to 6.4 x 2.0 to 3.3 µm (n=35). In addition, a brown discoloration was produced on malt extract agar (MEA) after using the 1 mol/L NaOH solution for 1 h. These characteristics were consistent with the description of Epicoccum sp. (Chen et al. 2017). To comfirm this identification, the internal transcribed spacer (ITS), large subunit ribosomal RNA (LSU), beta-tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) regions were amplified using the detailed primer pairs described by White et al., Rehner and Samuels, Woudenberg et al. and Liu et al., respectively. They had 99.8-100% homology to the ITS (GenBank no. MN215613, 504/505 bp), LSU (MN533800, 809/809 bp), TUB (MN329871, 333/333 bp), and RPB2 (MG787263, 596/596 bp) sequences of E. latusicollum in the GenBank database. A neighbor-joining phylogenetic tree was generated based on the concatenated sequences of all the above regions in MEGA7. The DZY3-3 clustered in the E. latusicollum clade with 100% bootstrap support. Koch's postulates were performed by spray inoculation (1×106 spores/mL) on the left sides of leaves of three healthy L. gracile seedlings and detached leaves, using isolate DZY3-3, while sterilized water served as the control was sprayed on the right sides of leaves. All plants and detached leaves were covered with clear polyethylene bags to maintain about 80% relative humidity at 25℃. Whether in vivo or in vitro pathogenicity test showed similar symptoms to those occurred in the field after 5 days post inoculation. No symptoms occurred on controls. The experiment was repeated three times. Subsequently, the same fungus was reisolated and identified from leaves of three inoculated seedlings. The E. latusicollum has a very wide host range. For example, it has been reported to cause stalk rot on Maize (Xu et al. 2022) and cause leaf spot on Tobacco in China (Guo et al. 2020). To our knowledge, it is the first report of E. latusicollum causing leaf spot on L. gracile in the world. This study will provide an important reference for the biology of E. latusicollum and the distribution of the disease.

Stem rot on Adenia globosa caused by Lasiodiplodia theobromae in Jiangsu.

Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China. In September 2022, a new stem basal rot disease was observed on A. globosa seedlings, being planted here. Stem basal rot were observed on approximately 80% of A. globosa seedlings. The basal stem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and ß-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 µL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch's postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) and root of Citrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.

First report of leaf spot on pecan (Carya illinoinensis) caused by Stemphylium eturmiunum in China.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important nut tree species, which has been widely planted in Jiangsu and Anhui Provinces of China in recent years (Mo et al. 2018). In May 2022, a new leaf spot disease was observed on both young and old leaves of pecan trees in the Luhe area, Nanjing, Jiangsu Province. Approximately 30% of pecan trees suffered from the disease, which affected the growth of young trees and nut production to cause economic loss. Initially, the leaf spots were grayish black and round. Then, disease spots enlarged and joined together, forming irregular lesions with uneven edges. In the last stage, the leaflets were withered. To isolate the pathogen, three symptomatic leaves were collected from each of three different pecan trees. Leaf sections (3 to 4 mm) were excised from the margin of spots, surface sterilized in 75% alcohol for 30 s, then sterilized in 1.5% NaClO for 90 s. After rinsing three times with sterile distilled water, leaf sections were placed on potato dextrose agar (PDA) medium and incubated at 25 °C in a dark environment for 5 days. Pure cultures were obtained by monosporic isolation. A total of 20 isolates were obtained, and 12 isolates were identified as Stemphylium sp. with the same morphological features and ITS sequences. A representative isolate, named LH3-3, was selected for further study. Colonies on PDA were light yellow with dense mycelium and were brownish yellow on the reverse side. Conidia were 16.3 to 34.4 × 8.1 to 16.3 µm) (n=35), muriform, brown, with transverse and longitudinal septa, lightly deformed at the transverse septa. Ascomata were not observed. The morphological characteristics were consistent with the description of Stemphylium eturmiunum (Simmons 2001). The internal transcribed spacer region (ITS) and portions of genes for calmodulin (cmdA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) were amplified and sequenced with the primers ITS1/ITS4 (White et al. 1990), CALDF1/CALDR2 (Xu et al. 2022) and GPD-F/R (Xie et al. 2019), respectively. Sequences were deposited in GenBank under accessions OP482492 (ITS), OP495734 (cmdA), and OP495735 (gpd). BLAST analysis showed that the sequences had 99.67-100% homology to ITS (525/525 bp) of S. eturmiunum strain ST14 (MH843733), cmdA (694/694 bp) of strain CBS122124 (KU850832), and gpd (299/300 bp) of isolate UMSe0030 (MK336876). MEGA 7.0 was used to construct a phylogenetic analysis based on concatenated sequences of ITS, cmdA, and gpd using the neighbor-joining method. The results showed that LH3-3 clustered on the branch of S. eturmiunum, and the support rate was 100%. A spore suspension in sterile water was made from strain LH3-3 grown on PDA, and adjusted to 1×106 spores/mL with a hemocytometer. To test pathogenicity, 20 µl drops of the spore suspension were placed on the left sides of four healthy detached leaflets of mature pecan trees and leaves of three 3-month-old seedlings. The right side of each leaflet was inoculated with 20 µl drops of sterile distilled water as the control. All inoculated seedlings and detached leaflets were covered with a transparent plastic bag and cultured in a greenhouse at 25 °C, 80% relative humidity, and a 12 h light cycle until symptom appeared. The experiment was repeated three times. After 7 days of inoculation, grayish black lesions appeared on all inoculation sites with the spore suspension but not in the controls. The leaf spot symptoms were similar to those observed in orchards. The same fungus, identified by morphological characteristics and sequencing of ITS, cmdA, and gpd, was re-isolated from the diseased spots of the inoculated leaflets to complete Koch's postulates. S. eturmiunum has been reported to infect garlic (Dumin et al. 2022) and tomato (Prencipee et al. 2021), but this is the first report of S. eturmiunum causing leaf spot of C. illinoinensis. This study provides a basis for further study on the biology, epidemiology, and management of the disease.

Luteolin is an Effective Component of Platycodon grandiflorus in Promoting Wound Healing in Rats with Cutaneous Scald Injury.

Background: Platycodon grandiflorus could significantly improve the pathological results of cutaneous scald injury, reduce the release of inflammatory factors and promote angiogenesis. This study investigated the wound healing effect of luteolin, an active component of P. grandiflorus, on induced cutaneous scald injury in Sprague-Dawley (SD) rats. Methods: The protein expression levels of TNF-α and IL-6 were detected by ELISA. QRT-PCR was adopted to detect the expression of TGF-ß1 and VEGF. Histopathological changes of scald wounds were analyzed by hematoxylin-eosin staining. Cell viability and migration ability were detected by CCK-8 assay and scratch assay. Results: Both in vivo and in vitro experiments showed that luteolin promoted wound healing of cutaneous scald injury. Gene Oncology (GO) functional analysis and rescue experiments showed that endothelial nitric oxide synthase 3 (NOS3) was the critical target of luteolin in treating cutaneous scald. Conclusion: This study demonstrated that luteolin is an effective component of P. grandiflorus and is effective in the treatment of cutaneous scald injury.

The key molecular pattern BxCDP1 of Bursaphelenchus xylophilus induces plant immunity and enhances plant defense response via two small peptide regions.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H2O2 production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana, which also induced the expression of pathogenesis-related (PR) genes (PtPR-3, PtPR-4, and PtPR-5) in P. thunbergii and enhanced the resistance of the host to B. xylophilus. These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii, and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future.

First report of leaf spot on Deutzia crenata caused by Neopestalotiopsis ellipsospora in China.

Deutzia crenata Sieb. et Zucc, native to Japan, with white flowers in early summer, is a high quality ornamental shrub widely planted in China. In October 2021, a new leaf spot disease was observed on approximately 70% of the 320 D. crenata trees growing in Nanjing Botanical Garden, Jiangsu Province, China. The disease started as irregular small gray spots on the leaf of D. crenata that coalesced into larger lesions. Infected leaves turned yellow (Figure S1A) and leaves with multiple spots withered. To isolate the pathogen, leaf sections (3 to 4 mm) were excised from the lesion margin, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate (L-1), growing on PDA was circular, white, and cottony, and the surface undulate and pale luteous (Figure S1B). The reverse was similar in color (Figure S1C). The conidial masses were black and appeared over PDA plates after 12 days (Figure S1D). Conidia [18.3 to 28.4×5.4 to 8.5 µm (mean 24.5×6.7 µm)] (n=35) were fusiform to ellipsoid and four-septate (one basal and one apical cell hyaline, and three brown median cells), with two to three apical appendages (Figure S1E). These characteristics were consistent with the description of Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Three regions of the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1α), and ß-tubulin (TUB) genes (GenBank Accession No. OM663738, No. OM687134 and No. OM687133, respectively) were amplified and sequenced with the primers pairs ITS1/ITS4 (Innis et al. 1990), EF1-526F/EF1-1567R (Maharachchikumbura et al. 2014) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were 95.4-99.8% similar to those from Neopestalotiopsis sp. accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate L-1 clustered in the N. ellipsospora clade with 98% bootstrap support (Figure S2). To test pathogenicity, three detached healthy leaves and three one-year-old D. crenata seedlings were inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. The right side of each leaf was inoculation with 20 µL of sterile water as the experimental control. All plants were covered with clear polyethylene bags and incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, leaf spots typical of those observed in the orchards were observed on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1F-G). The same fungus was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates (Figure S1H). N. ellipsospora is known to cause leaf spots on Camellia sinensis and sweet potato, infects fruits of Ardisia crenata in China (Maharachchikumbura et al. 2014; Maharachchikumbura et al. 2016; Wang et al. 2019), and causes stem spots on Acanthopanax divaricatus in Korea (Yun et al. 2015). This is the first report of N. ellipsospora causing leaf spot on D. crenata in the world. The occurrence of this disease needs to be monitored, because it can reduce the ornamental value of D. crenata. This finding provides the foundation to further investigate the biology and epidemiology of this disease so that effective strategies can be developed to manage this disease.

First report of leaf spot on Magnolia grandiflora caused by Lasiodiplodia theobromae in Jiangsu.

Magnolia grandiflora linn, with large and fragrant flowers, is widely planted in the south of Yangtze River valley in China. It is an excellent street tree and a beautiful ornamental tree for landscaping. In October 2021, a new leaf spot disease was observed on M. grandiflora seedlings and mature trees growing in Nanjing Botanical Garden, Jiangsu Province, China. According to statistics, about 300 M. grandiflora trees were planted here, and approximately 60% of M. grandiflora trees suffered from the disease. In the beginning, small black spots appeared on the leaf of M. grandiflora, and then the disease spots were connected into coalesced, and eventually lead to a large area of leaf dead (Figure S1A). To isolate the pathogen, ten diseased leaves were collected from ten plants distributed in different five areas of the botanical garden. The leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. Twenty-three isolates were obtained (the isolate rate of 72%), and identified as Lasiodiplodia sp.. A representative isolate, G-H-1 was used for the further study. The colony of G-H-1, growing on PDA was cotton-like. The primary mycelia was gray and white in the early stage of culture. It gradually turned black gray in the later stage, and the reverse was similar in color (Figure S1B). The pycnidia (fruiting body) was black and appeared over PDA plates after 15 days (Figure S1C). The hyphae of G-H-1 were dark brown, and the conidia were monospora, oval or elliptic, with a size of (9.6 ~ 13.3) µm× (5.7 ~ 8.0) µm (mean 11.7×6.6 µm, n=35) (Figure S1D). In the pycnidia, the conidiophores were inside and produced conidia (Figure S1E). In the early stage, the conidia of G-H-1 were colorless transparent, then gradually turned dark brown with a septum in the center (Figure S1F). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The regions of ITS, translation elongation factor 1-alpha (TEF1α) and ß-tubulin (TUB) genes (GenBank Accession No.OM698339, No.OM942757, and No.OM942756, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995). The obtained sequences showed 99.05-99.81% similarity with those from L. theobromae accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate G-H-1 clustered in the L. theobromae clade with 96% bootstrap support (Figure S2). To test pathogenicity, three one-year-old M. grandiflora seedlings that previously had been wounded with a sterile needle were inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation with 20 µL sterile water was treated as the control, which were inoculated on the right sides of leaves. All plants were covered with clear polyethylene bags to keep moisture. And inoculated detached leaves were incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical black spots were found on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1G-H). After 25 days of inoculation, perforation occurred at the black spots on the leaves of the inoculated plants, resulting in incomplete leaf (Figure S1I), which is identical disease symptoms to those observed in garden. The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates. The pathogen has a very wide host range. For example, it has been reported to cause dieback and sooty canker on Ficus trees (Abo Rehab et al. 2014), infected trunk of sultana seedless (Tang et al. 2021) and castor bean (Akgul et al. 2015), root of Citrus (Al-Sadi et al. 2014), date palm, and Mango (Al-Sadi et al. 2013) and Cassia fistula (Deng et al. 2015). But, according to nt.ars-grin.gov, there are no other reports of L. theobromae on M. grandiflora in the world. So, this would be the first one. This study provides an important reference for the biology, epidemiology.

A Bursaphelenchus xylophilus Effector, BxSCD3, Suppresses Plant Defense and Contributes to Virulence.

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus. BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in Pinus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

The Bursaphelenchus xylophilus effector BxML1 targets the cyclophilin protein (CyP) to promote parasitism and virulence in pine.

BACKGROUND: Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD) that has caused enormous ecological and economic losses in China. The mechanism in the interaction between nematodes and pine remains unclear. Plant parasitic nematodes (PPNs) secrete effectors into host plant tissues. However, it is poorly studied that role of effector in the infection of pine wood nematode (PWN). RESULTS: We cloned, characterized and functionally validated the B. xylophilus effector BxML1, containing an MD-2-related lipid-recognition (ML) domain. This protein inhibits immune responses triggered by the molecular pattern BxCDP1 of B. xylophilus. An insitu hybridization assay demonstrated that BxML1 was expressed mainly in the dorsal glands and intestine of B. xylophilus. Subcellular localization analysis showed the presence of BxML1 in the cytoplasm and nucleus. Furthermore, number of B. xylophilus and morbidity of pine were significantly reduced in Pinus thunbergii infected with B. xylophilus when BxML was silenced. Using yeast two-hybrid (Y2H) and coimmunoprecipitation (CoIP) assays, we found that the BxML1 interacts with cyclophilin protein PtCyP1 in P. thunbergii. CONCLUSIONS: This study illustrated that BxML1 plays a critical role in the B. xylophilus-plant interaction and virulence of B. xylophilus.

A novel pine wood nematode effector, BxSCD1, suppresses plant immunity and interacts with an ethylene-forming enzyme in pine.

The plant-parasitic nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), causes enormous economic loss every year. Currently, little is known about the pathogenic mechanisms of PWD. Several effectors have been identified in B. xylophilus, but their functions and host targets have yet to be elucidated. Here, we demonstrated that BxSCD1 suppresses cell death and inhibits B. xylophilus PAMP BxCDP1-triggered immunity in Nicotiana benthamiana and Pinus thunbergii. BxSCD1 was transcriptionally upregulated in the early stage of B. xylophilus infection. In situ hybridization experiments showed that BxSCD1 was specifically expressed in the dorsal glands and intestine. Cysteine residues are essential for the function of BxSCD1. Transient expression of BxSCD1 in N. benthamiana revealed that it was primarily targeted to the cytoplasm and nucleus. The morbidity was significantly reduced in P. thunbergii infected with B. xylophilus when BxSCD1 was silenced. We identified 1-aminocyclopropane-1-carboxylate oxidase 1, the actual ethylene-forming enzyme, as a host target of BxSCD1 by yeast two-hybrid and coimmunoprecipitation. Overall, this study illustrated that BxSCD1 played a critical role in the B. xylophilus-plant interaction.

Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees.

BACKGROUND: The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. RESULTS: In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. CONCLUSIONS: The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

BxCDP1 from the pine wood nematode Bursaphelenchus xylophilus is recognized as a novel molecular pattern.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which causes serious damage to pine forests in China. Plant immunity plays an important role in plant resistance to multiple pathogens. Activation of the plant immune system is generally determined by immune receptors, including plant pattern recognition receptors, which mediate pattern recognition. However, little is known about molecular pattern recognition in the interaction between pines and B. xylophilus. Based on the B. xylophilus transcriptome at the early stages of infection and Agrobacterium tumefaciens-mediated transient expression and infiltration of recombinant proteins produced by Pichia pastoris in many plant species, a novel molecular pattern (BxCDP1) was characterized in B. xylophilus. We found that BxCDP1 was highly up-regulated at the early infection stages of B. xylophilus, and was similar to a protein in Pararhizobium haloflavum. BxCDP1 triggered cell death in Nicotiana benthamiana when secreted into the apoplast, and this effect was dependent on brassinosteroid-insensitive 1-associated kinase 1, but independent of suppressor of BIR1-1. BxCDP1 also exhibited cell death-inducing activity in pine, Arabidopsis, tomato, pepper, and lettuce. BxCDP1 triggered reactive oxygen species production and the expression of PAMP-triggered immunity marker genes (NbAcre31, NbPTI5, and NbCyp71D20) in N. benthamiana. It also induced the expression of pathogenesis-related genes (PtPR-3, PtPR-4, and PtPR-5) in Pinus thunbergii. These results suggest that as a new B. xylophilus molecular pattern, BxCDP1 can not only be recognized by many plant species, but also triggers innate immunity in N. benthamiana and defence responses of P. thunbergii.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

An Effector, BxSapB1, Induces Cell Death and Contributes to Virulence in the Pine Wood Nematode Bursaphelenchus xylophilus.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

[Effect of NH4(+) -N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism and main chemical composition of Pinellia ternata].

OBJECTIVE: To study the effect of nitrogen forms on nitrogen metabolism and main chemical composition of Pinellia ternate. METHOD: Through the soilless cultivation experiment and based at the same nitrogen level and different NH4(+) -N/NO3(-) -N ratios, nitrate reductase (NR) activity, glutamine synthetase (GS) activity, the content of nitrate nitrogen and ammonium nitrogen in different parts of P. ternate were determined. The contents of total alkaloid, free total organic acids and guanosine in the tuber were determined. The yield of bulbil and tuber was calculated. RESULT: The test results showed that, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of nitrate reductase decreased, the content of nitrate nitrogen in the leaves, petioles and tuber increasing initially, then decreased, and the content of nitrate nitrogen in the root decreased. Meanwhile, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of glutamine synthetase in the leaves, petioles and root increased, the activity of glutamine synthetase in the tuber increasing initially, then decreased. The contents of ammonium nitrogen in the leaves, tuber and root increased initially, then decreased, and the contents of ammonium nitrogen in the petioles increased with the NH4(+)(-N/NO3(-)-N ratio increasing. The yield of bulbil and tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 75: 25. The content of total alkaloid and guanosine in the tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 0: 100, and the contents were 0.245% and 0.0197% respectively. With the NH4(+)-N/NO3(-) -N ratio of 50: 50, the content of free total organic acids was the highest, it reached 0.7%, however, the content of free total organic acids was the lowest at the NH4(+) -N/NO3(-) -N ratio of 0: 100. CONCLUSION: Nitrogen fertilization significant influences the nitrogen metabolism, the yield and main chemical composition of P. ternate.

[Dynamic accumulation of dry matter and active element in Pinellia ternata of Taizhou].

OBJECTIVE: To study the dynamic accumulation of dry matter and index components and provide reference to the manual cultural technique of Pinellia ternata. METHOD: Seedlings of Taizhou P. ternata were used as the pot experiment material, and the contents of free total organic acid and guanosine of each organ were determined at different stages of spring and autumn period. RESULT: P. ternata had two growth climax in spring and autumn. Under the same condition of cultivation and management, dry matter largely accumulated in spring. The contents of free total organic acid and guanosine was ascending during the growth period, but dropped during sprout tumble bolting and sprout tumble stage. CONCLUSION: High temperature and bolting affect the accumulation of organic acids significantly, it is reasonable to prevent the temperature stress in production.