Insights of the Neofusicoccum parvum–Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch’s postulates—followed by a molecular approach—revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue–N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction.

First Field Record of the Tropical Red-Banded Thrips Selenothrips rubrocinctus (Thripidae: Panchaetothripinae) in Europe

Red-banded thrips Selenothrips rubrocinctus is a polyphagous pest widely distributed in tropical and subtropical regions. Various fruit trees such as cacao, cashew, mango, avocado, and guava in certain habitats are attacked. This is the first report of the spontaneous occurrence of outdoor populations of this species of thrips in Europe. This species has been found in northern Italy on Liquidambar styraciflua, Carpinus betulus, and other ornamental forest species in urban areas. Barcode sequences of the Italian specimens were obtained.

First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch’s postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

First records of Xylosandrus compactus (Coleoptera: Curculionidae, Scolytinae) in the Iberian Peninsula: an expanding alien species?

Xylosandrus compactus (Eichhoff) (Col.: Curculionidae, Scolytinae) is an ambrosia beetle species native to subtropical Eastern Asia, with great concern due to its high invasive ability. This species has invaded 54 countries worldwide, including 4 European countries (Italy, France, Greece, and Spain); it was detected in Mallorca (Balearic Islands, Spain) in October 2019. In the present work, X. compactus is recorded for the first time in the Iberian Peninsula (Girona province, NE Spain); specimens were collected in Banyoles (August 2020, attacking twigs of Laurus nobilis and Liquidambar styraciflua) and Platja d’Aro (October 2020, attacking twigs of L. nobilis). Up-to-date information is presented about its geographical distribution, host plants, biology, symptoms, associate damages, and the possible origin of this species in Europe.

First report of leaf spot disease caused by Stemphylium eturmiunum on American sweetgum (Liquidambar styraciflua L.) in China

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23–35 × 6–10 µm) (n=60) were solitary, straight to flexuous. Conidia (19–34 × 10–21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch’s postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

First molecular insights into the infection process provoked by Neofusicoccum parvum in Liquidambar styraciflua and the identification of new cysteine-rich proteins in both organisms

Background Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which groups endophytic and latent pathogens of woody plants responsible for diseases such as cankers, dieback and blight. It is a widespread pathogen with a broad host range, including agricultural, horticultural and forestry plants; therefore, it is relevant to characterize the molecular mechanisms involved in the disease caused by this pathogen. This work reports for the first time N. parvum as a pathogen of Liquidambar styraciflua. We established an in vitro pathosystem using foliar tissue to characterize the infection process through scanning electron microscopy (SEM). Because cysteine-rich proteins (CysRPs) have been studied for their important functions in plant-pathogen interactions, new CysRPs were identified for these organisms, and mRNA expression of these proteins was analyzed at early time points during the interaction.Results 24 hours post infection, the pathogen caused visible symptoms, and microscopic analysis at 16 days post infection revealed the presence of N. parvum pycnidia embedded in L. styraciflua leaf tissue. For both organisms, two databases with transcriptomic and genomic information were analyzed, and five new CysRPs were identified for each organism. The length varied between 95 and 204 amino acids, and in silico analysis revealed that all the proteins are potentially secreted. The search for conserved domains and phylogenetic analyses revealed that all the proteins are novel, including two of N. parvum that present the well-known CFEM domain. RT-qPCR analysis was conducted at 24 and 72 hours post infection, and the results showed changes in the levels of CysRP mRNAs for both the plant and the fungus at early stages during the interaction.Conclusions N. parvum was identified for the first time as a pathogen of L. styraciflua, and this work presents an approach to comprehensively understand the molecular mechanisms involved in this interaction, highlighting the potential involvement of CysRPs of both organisms under this biotic stress.

Colletotrichum spp. causing anthracnose on ornamental plants in northern Italy

Species of Colletotrichum are considered among the most important plant pathogens, saprobes and endophytes on a wide range of ornamentals, fruits and vegetables. Several Colletotrichum species have been reported in nurseries and public or private gardens in northern Italy. In this study, the occurrence, diversity and pathogenicity of Colletotrichum spp. associated with several ornamental hosts was explored. Survey were carried out during the 2013–2019 period in Piedmont, Italy. A total of 22 Colletotrichum isolates were collected from symptomatic leaves and stems of two Campanula spp., Ceanothus thyrsiflorus, Coreopsis lanceolata, Cyclamen persicum, Hydrangea paniculata, Liquidambar styraciflua, Mahonia aquifolium and Rhyncospermum jasminoides. A multi-locus phylogeny was established based on the basis of three genomic loci (gapdh, act and tub2). The pathogenicity of selected, representative isolates was tested. Colletotrichum isolates were identified as members of four important species complexes: Acutatum, Gloeosporioides, Dematium and Destructivum. Colletotrichum fioriniae, C. nymphaeae and C. fuscum were found in association with leaf lesions of Mahonia aquifolium, Campanula rapunculoides and Coreopsis lanceolata, respectively. Colletotrichum lineola, C. grossum and C. cigarro were isolated from Campanula trachelium, Rhyncospermum jasminoides and Liquidambar styraciflua, respectively. Colletotrichum fructicola was found to be responsible of anthracnose of Ceanothus thyrsiflorus, Hydrangea paniculata, Cyclamen persicum and Liquidambar styraciflua. All the tested isolates were pathogenic and reproduced identical symptoms to those observed in private gardens and nurseries. The present study improves our understanding of Colletotrichum spp. associated with different ornamental hosts and provides useful information for an effective disease management programme.