First Report of Fusarium avenaceum Causing Canker Disease on Kiwi Tree in China

In May 2021, canker symptoms were detected on ‘Xuxiang’ kiwi trees in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Seven-year-old trees exhibited black necrotic lesions and cracked areas in the trunk (Figure 1). The symptoms were observed in approximately 10% of the trees in 6 orchards (31 ha in total). Application of commercial fungicides did not control the advancement of the pathogen, and infected trees were removed to control the spread. Three samples, approximately 1 cm2 in size, of symptomatic tissue were collected and surface sterilized in 2% NaOCl for 1 min, and washed with sterile ddH2O. Four isolates showing white mycelium with yellow pigmentation were obtained after 4 days of incubation on PDA, containing chloramphenicol (50 µg/mL), at 28 ºC. The pathogen was isolated from all collected samples. ITS, EF1-α, TUB2, RPB1 and RPB2 genes were amplified using ITS1/ITS4, EF1-728F/EF1-986R, T1/T22, RPB1-5F/RPB1-8R and RPB2-5F/RPB2-7cR (strain NJC06), or RPB2-c7F/RPB2-11aR (strains NJC07 and NJC08), primers, respectively. Two isolates shared the same sequences (strain NJC08). Obtained sequences were submitted to GenBank under accession numbers MZ669205 and OL347898-OL347899 (ITS), OL439731-OL439733 (EF1-α), OL439734-OL439736 (TUB2), OL439737-OL439739 (RPB1), and OL439740-OL439742 (RPB2). The sequences shared >99% (ITS; F. avenaceum CBS 128538, MH864972), >99% (EF1-α; F. avenaceum 55-2, MN473124), 100% (TUB2; F. avenaceum SICAUCC 18-0001, MK253102), >98% (RPB1; F. avenaceum NRRL 26911, MG282372), and >98% (RPB2; F. avenaceum SICAUCC 18-0001, MK396098; or F. avenaceum FRC R-09495, CQ915486) homology to multiple F. avenaceum strains. Molecular phylogenetic tree (Figure 2) was constructed using MEGA7 with Fusarium strains found causing rot in various hosts (Wang et al. 2015), and other fungal species, such as Cadophora nalorum, Diaporthe ambigua, D. australafricana, and Neofusicoccum parvum, which were reported to cause cordon dieback on kiwi tree in Chile (Diaz et al. 2021). Microscope observations after cultivation of all isolates on barley-honey-tryptone medium (Song et al. 2020) showed the presence of septate mycelium, fusiform microconidia (8-15 µm in length, containing between 0 and 3 septa; n = 77) and chlamydospores (n = 21), and agree with the morphology of F. avenaceum (Zhao et al. 2020). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the trunk of 3-months-old ‘Xuxiang’ kiwi trees. Solutions containing 1 × 106 spores/mL (20 µL) of the isolates were injected in the wounds. Sterile ddH2O was used for the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 80% relative humidity for 4 days. The pathogen was recovered from the canker lesions, which were similar to those observed in the orchards, and its identity was confirmed by sequence analysis. The pathogen only infected wounded trees, and probably invaded the orchards during the pruning in February 2021. F. avenaceum was reported to cause canker on almond tree (Stack et al. 2020), stem rot on Anthoxanthum aristatum and Polygonatum cyrtonema (Pieczul et al. 2018; Xu et al. 2019), and root rot on carrot, Coptis chinensis and wheat (Le Moullec-Rieu et al. 2020; Mei et al. 2020; Ozer et al. 2020). Recently, F. avenaceum was found causing fruit blotch in kiwi fruit in Anhui (China) (Zhao et al. 2020). Here, F. avenaceum was found causing canker disease in kiwi tree, demonstrating the host and tissue promiscuity of this pathogen. Kiwi is an important crop in China with nearly 1.5 million tons produced in 2019. This report will help to better understand the pathogens reducing kiwi production in China.

First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

Identification and Characterization of Nectria pseudotrichia Associated with Camellia Canker Disease in China

Camellia japonica is a native tree species with high economic value that is widely cultivated in southern China. In recent years, canker disease has been observed in camellia plantations in Zhejiang Province, China, with the disease incidence rate in some plantations exceeding 20%. Canker disease severely affects the trunks and branches of C. japonica in China, but the causal agent has not yet been identified. In this study, the pathogen was isolated from infected C. japonica tissues through a conventional tissue isolation approach. Species identification was conducted using morphological methods combined with multilocus phylogenetic analysis. Pathogenicity was tested based on Koch’s postulates. The results showed that the pathogen could be isolated from the diseased bark of C. japonica ‘Hongluzhen’. The pathogen was identified as Nectria pseudotrichia based on morphological, cultural, and molecular traits. The inoculation of the pathogen into C. japonica ‘Hongluzhen’ caused necrotic lesions on healthy seedlings, and the fungus N. pseudotrichia could be re-isolated from such lesions. Therefore, N. pseudotrichia is the causal agent of canker disease affecting C. japonica in China.

Response of tomato (Solanum lycopersicum) varieties to Clavibacter michiganensis subsp. michiganensis infection

<p><em>Clavibacter michiganensis</em> subsp. <em>michiganensis</em> causes bacterial wilt and canker disease of tomato. The objectives of this research were i) to characterize and identify the most aggressive Cmm in Chignahuapan, Puebla, ii) to evaluate the tolerance in two phenological stages of 10 tomato varieties to infection by CP_Cmm1 in the greenhouse and the <em>in vitro</em> sensitivity of the bacteria to bactericides. 12 Cmm isolates were collected from tomato plants and a strain (CP_Cmm-1) with greater aggressiveness was selected according to the time of manifestation of symptoms in plants of var. Reserva. Afterward, the strain was inoculated by cutting with scissors embedded in a suspension with 3 x 108 UFC mL-1 in two phenological stages (at five and 10 true leaves). Symptoms were evaluated for 30 days after inoculation. Additionally, the sensitivity of CP_Cmm-1 to eight commercial bactericides <em>in vitro</em> was evaluated. The results showed that the CP_Cmm-1 strain was identified with 97% similarity with the API20 E system, positive with DAS-ELISA, and by PCR it showed 96.6% identity with <em>Clavibacter michiganensis</em> subsp. <em>michiganensis</em>. The Saher variety showed greater tolerance to infection (* = p?0.05) both in the stage of five and 10 leaves; the varieties Sv4401, Nápoles, and Súper óptimo showed greater susceptibility in the five-leaf stage. The Sv4401 variety was highly susceptible in both stages. Neither variety was resistant to infection by CP_Cmm-1. The use of the Sahel variety and kasugamycin could reduce the damage of this bacteria.</p>

Phylogenetic analysis shows that New Zealand isolates of Neonectria ditissima are similar to European isolates

Neonectria ditissima causes a debilitating apple tree canker disease. We determined the efficacy of polymerase chain reaction primers, originally designed for European strains, by sequencing New Zealand strains. The concatenated ribosomal inter-transcribed spacer and β-tubulin gene regions of 17 New Zealand isolates were compared with those of two European strains by phylogenetic analysis. New Zealand and European isolates of N. ditissima were in the same clade, suggesting that there has been little change in these gene regions following introduction to New Zealand. There was 100% homology with Bt-FW135 and Bt-RW284 primers. Based on sequencing 17 New Zealand isolates from several locations, these polymerase chain reaction primers can be relied upon to amplify New Zealand isolates of N. ditissima.

Genetic diversity and potential inoculum sources of Fusarium species causing cankers in bareroot-propagated almond trees in California nurseries

Previous research determined that Fusarium acuminatum and Fusarium avenaceum are important causal agents of a canker disease in bareroot-propagated fruit and nut trees in California that emerges during cold-storage or after transplanting. The disease largely disappeared after 2001, but it reemerged in 2011 in almond trees in at least one nursery. This motivated further study of the etiology and epidemiology of the disease by undertaking studies to determine distribution of the pathogens throughout almond nursery propagation systems and trace possible sources of inoculum. Research initiated in 2013 detected pathogenic Fusarium spp. throughout the almond propagation system, including in healthy trees, in soils, on wheat rotation crops, on equipment, and in the cold storage facility air. In addition to the two Fusarium spp. implicated previously, Fusarium brachygibbosum and a new Fusarium species, Fusarium californicum, were found to be pathogenic on almond trees. Multi-locus sequence typing and somatic compatibility testing confirmed that isolates within a species collected from different materials in the nursery were all highly genetically similar and likely of one clonal lineage. These findings affirm that equipment surfaces, wheat rotation crops, soil, cold storage facility air, and asymptomatic almond tree materials (i.e., rootstock cuttings, budwood, and scions) can potentially contribute inoculum to increase disease prevalence and severity.

Influence of Trichoderma spp on Macrophoma theicola branch canker disease in South Indian tea gardens, India

Branch canker disease caused by the fungus Macrophoma theicola is a major stem disease that reduces the yield of south Indian tea plantations. Hence the present study aimed to assess the efficacy of the biocontrol agent Trichoderma spp against various isolates of Macrophoma spp. For this matter, different tea-growing regions of south India were surveyed for the isolation and characterization of Macrophoma spp. Then, fungal biocontrol strains (Trichoderma viride, Trichoderma atroviride, Trichoderma harzianum, and Gliocladium virens) were procured from microbial type culture collection Centre (MTCC) to screen their antagonistic potential on different isolates Macrophoma spp. The spores of Macrophoma spp were examined through a light microscope and identified by their peculiar morphological features such as non-septum pycnidiospores present in the sac and oval shape spore with stalk and confirmed using 18S rRNA gene sequence. The results revealed that the biocontrol G. virens followed by T. harzianum showed a higher inhibitory effect on different isolates of Macrophoma spp in the dual plate and culture filtrate studies. In the well diffusion method, the fungal biocontrol agents were found to be exhibit non-significant differences on different isolates of branch canker pathogen. The hyphal interactions studies showed that the pathogenic hyphal wall shrunk and penetrated by the interaction of G. virens.

Effects of Phytophthora Inoculations on Photosynthetic Behaviour and Induced Defence Responses of Plant Volatiles in Field-Grown Hybrid Poplar Tolerant to Bark Canker Disease

Bark cankers accompanied by symptoms of decline and dieback are the result of a destructive disease caused by Phytophthora infections in woody plants. Pathogenicity, gas exchange, chlorophyll a fluorescence, and volatile responses to P. cactorum and P. plurivora inoculations were studied in field-grown 10-year-old hybrid poplar plants. The most stressful effects of P. cactorum on photosynthetic behaviour were found at days 30 and 38 post-inoculation (p.-i.), whereas major disturbances induced by P. plurivora were identified at day 30 p.-i. and also belatedly at day 52 p.-i. The spectrum of volatile organic compounds emitted at day 98 p.-i. was richer than that at day 9 p.-i, and the emissions of both sesquiterpenes α-cubebene and germacrene D were induced solely by the Phytophthora inoculations. Significant positive relationships were found between both the axial and the tangential development of bark cankers and the emissions of α-cubebene and β-caryophyllene, respectively. These results show that both α-cubebene and germacrene D are signal molecules for the suppression of Phytophthora hyphae spread from necrotic sites of the bark to healthy living tissues. Four years following inoculations, for the majority of the inoculated plants, the callus tissue had already closed over the bark cankers.

High-quality genome assembly and annotation resource of Botryosphaeria dothidea strain BDLA16-7, causing trunk canker disease on Chinese hickory

Botryosphaeria dothidea is a latent pathogen with global importance to woody plant health, which causes serious tree trunk cankers on Chinese hickory. To date, only one Illumina short-read-based genome assembly of strain CK16 is available for host Chinese hickory. To address this problem, we reported a near telomere-to-telomere genome assembly of strain BDLA16-7 (46.05 Mb, N50 3.87 Mb) using Oxford Nanopore Sequencing Technology. Our genome assembly was consisted of 15 contigs, of which, 3 were assembled into chromosomal level and the maximum contig length was 6.19 Mb. The assembly contained 7.96% repeats and 12,815 protein-coding genes (10,274 genes were functional annotated). We also identified 3,642 pathogen-host interaction (PHI) genes, 250 carbohydrateactive enzymes (CAZymes), 252 cytochrome P450 enzymes (CYPs), 752 putative secreted proteins and 63 secondary metabolite biosynthesis gene clusters (SMBGCs). The BUSCO completeness of genome assembly and predicted genes was 99.34% and 97.50%, respectively, at fungal level (n=758). The almost chromosomal-level and well-annotated genome assembly will provide a valuable genetic resource for understanding of the infection mechanisms of B. dothidea in future.