First report of Fusarium proliferatum causing leaf spot on tea plants in Jiangxi Province, China.

Tea (Camellia sinensis (L.) O. Kuntze), a perennial evergreen shrub, is one of the most important cash crops in China. In September 2021, leaf spot symptoms were observed on approximately 30% of tea plants in a 2 ha commercial field of Lushan (29°33'0" N, 115°58'48" E), Jiangxi Province, China. The symptoms initially appeared as small, gray lesions, and later became larger (10-15 mm in diameter) circular to irregular spots with light brown centers and gray borders. To isolate the pathogen, small pieces (3×3 mm) cut from the margins of lesions were sterilized with 75% ethanol for 10 s, 0.1% HgCl2 for 20 s, and then rinsed three times with sterile water. The pieces were placed onto acidified potato dextrose agar (APDA) plates, and incubated in darkness at 28℃. Pure cultures were prepared by subculturing hyphal tips. A total of 16 fungal isolates were obtained, and the colonies of 15 isolates (isolation rate 93.8%) looked identical, resembling those of the genus Fusarium. The colonies were white to pink with purple woolly mycelium. After 10 to 15 days incubation, slightly curved macroconidia with three to four septa measuring 14.0 to 34.5 × 2.0 to 3.5 µm (n = 50), and oval, unicellar microconidia measuring 4.0 to 9.0 × 1.5 to 3.5 µm (n = 50) were observed. These morphological characteristics were similar to that described for Fusarium proliferatum (Leslie and Summerell 2006). Genomic DNA of representative isolates (LSZWY, LSZWY2, LSZWY3) was extracted with the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd, Shanghai). The translation elongation factor 1 alpha gene (EF-1ɑ) was amplified using primers EF-1H / EF-2T (O'Donnell, et al. 2015). PCR product was sequenced and the sequence was 709 bp (Accession No. OL614004, ON357634, ON595710). BLAST search results showed that it had 99.9% identity with the EF-1ɑ gene sequence of F. proliferatum (MH341215, MT371378). To test pathogenicity, nine leaves from 5-year-old healthy tea plants (Ca. Luyun 3) were wounded using a sterilized needle and inoculated with a 20µl conidial suspension (2 × 107 conidia·mL-1) on one side of the plants and the other side with sterilized distilled water as a control. All leaves were incubated in a growth chamber at 28℃ and 80% relative humidity with a 12 h light/dark photoperiod. Seven days later, all inoculated treatments showed symptoms identical to those observed in the field, while the control remained asymptomatic. The experiment was repeated three times with similar results. Koch's postulates were fulfilled by successful re-isolation and morphological and molecular identification of F. proliferatum from the inoculated leaves. This pathogen can cause diseases of many crops, e.g. tobacco, Polygonatum cyrtonema and others (Li, et al 2017; Zhou, et al. 2021). However, this is the first report of F. proliferatum causing leaf spot on tea plants in China. This new disease poses a threat to the yield and quality of tea and methods need to be developed for its control and to prevent further spread.

First Report of Colletotrichum liriopes Causing Leaf Spot of Rohdea japonica in China.

Rohdea japonica, known as Japanese sacred lily, a perennial herb in the family Asparagaceae, is an important ornamental plant in China (Hinkley 2006). In May 2022, at the campus of Jiangxi Agricultural University (28o45'50″N, 115o50'2″E), Nanchang, China, leaf spots occurred on R. japonica with a disease incidence of 95% (285/300 plants). Disease severity ranged from 40-60% of the leaf area on each plant. Symptoms initially appeared as small water-soaked spots, usually at the edges of the leaves. Then, the spots enlarged rapidly and became yellow brown, circular or semicircular. Finally, the dead tissues fell out of the leaf, leaving ragged leaf edges. Small pieces (3×3 mm) cut from the margin of necrotic leaf tissue were surface disinfested in 75% ethanol for 10 s and 0.1% HgCl2 for 20 s. After being rinsed three times with sterile distilled water, tissue was placed on potato dextrose agar (PDA) plates and incubated at 28℃ with a 12 h light-dark cycle. The growing fungal colonies were purified by subculturing hyphal tips, and 12 fungal isolates with similar morphology were obtained. Colonies were flat, smoke-grey with olivaceous green fan-shaped stripes. Conidia were single-celled, hyaline, slightly curved, both ends gradually tapering, measuring 14.9 - 25.3 µm long × 3.9 - 6.2 µm wide (n = 100). Appressoria were solitary, irregular, sometimes ellipsoidal, dark brown, measuring 6.5 - 17.6 µm long × 3.9 - 8.7 µm wide (n = 50). Morphological and cultural characteristics of the isolates matched the descriptions of Colletotrichum liriopes (Damm et al. 2009; Yang et al. 2020). To confirm the pathogens identity, genomic DNA of a representative isolate (WNQ1) was extracted, and the rDNA-ITS, TUB2 and CAL gene were amplified and sequenced using the primers ITS1/ITS4, T1/Bt2b, and CL1C/CL2C (White et al. 1990; Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession number: ON514224 (rDNA-ITS), ON552548 (TUB2) and ON552549 (CAL). BLAST analyses showed 100%, 99.73% and 100% identity with 100% query coverage to the rDNA-ITS, TUB2 and CAL sequence of C. liriopes (MK644098, HM585414 and MN803417, respectively). Phylogenetic analysis using concatenated sequences of rDNA-ITS, TUB2 and CAL placed the isolate WNQ1 in a single clade with the reference strain of C. liriopes CORCK2. To confirm pathogenicity, a conidial suspension (1×106 conidia/ml) of isolate WNQ1 was sprayed on three leaves each of three healthy R. japonica plants wounded with a sterile needle, whereas control plants were wounded in the same way and sprayed with sterile distilled water. All treated plants were placed in a moist incubator at 28℃ with a 12 h photoperiod. Three days later symptoms, similar to those described above appeared. Symptoms did not develop on the control leaves. C. liriopes was recovered from the inoculated leaves, fulfilling Koch's postulates. Anthracnose caused by C. liriopes in R. japonica plants has been reported in the United States and Korea. To our knowledge, this is the first report of anthracnose on R. japonica caused by C. liriopes in China. The disease seriously affected the ornamental value of R. japonica. The result provides the foundation to study the occurrence patterns and control measures of R. japonica anthracnose.

First Report of Fusarium oxysporum and F. proliferatum Causing Postharvest Corm Rot on Taro in China.

Taro (Colocasia esculenta), a perennial tuberous herb of the family Araceae, is cultivated widely in southern China. In December 2020, postharvest corm rot occurred on taro of 5 tons with approximately 70% incidence in a 18 square meter cellar in the Qingshanhu District (115°83'E, 28°76'N) of Nanchang City, Jiangxi Province, China. Infected corms had round, soft and slightly sunken lesions covered with white mycelia. The lesions gradually expanded, causing part or whole corm to become soft and shrink, and the inner corm tissue turned brown and rotten. To isolate the pathogen, a total of 30 diseased corm samples were collected. The corms were surface-disinfected by wiping them with 70% ethanol and then passing them over flame back and forth for 5 s. After epidermal tissue of the corms was removed using a sterilized scalpel, small portions of the inner tissue were transferred onto potato dextrose agar (PDA) and incubated at 25°C in the dark. A total of 27 isolates forming Fusarium-like colonies were obtained using monosporic isolation, of which 11 isolates were identified as F. oxysporum and 16 isolates were identified as F. proliferatum based on the colony characteristics and conidial morphology (Leslie and Summerell, 2006). Colonies of F. oxysporum isolates produced dense whitish to light purple mycelia with dark red pigments. Macroconidia were sickle-shaped, straight to slightly curved, 3-5 septa, measuring 25.6 to 45.8 × 3.3 to 6.1 µm. Microconidia were hyaline, oval or ellipsoid, aseptate, and measured 5.2 to 11.8 × 2.2 to 3.5 µm. Chlamydospores were round, 3.5 to 7.6 µm in diameter. Colonies of F. proliferatum isolates were whitish with abundant aerial mycelia and orange pigments. Numerous oval unicellular microconidia were 4.5 to 11.8 × 1.9 to 4.2 µm, and sparse falcate macroconidia with 3-4 septa were 19.4 to 39.2 × 1.9 to 5.2 µm in size. No chlamydospores were observed. Genomic DNA of two representative isolates (F. oxysporum isolate YTU1 and F. proliferatum isolate YTH1) was extracted, and the internal transcribed spacer (ITS) region and translation elongation factor 1-α (TEF1-α) gene were amplified and sequenced using primers ITS1/ITS4 and EF-1H/EF-2T (White et al., 1990; Zhang et al., 2014) respectively. Using BLAST analysis, the ITS sequences of isolates YTU1 (506 bp) and YTH1 (508 bp) exhibited 100% homology with F. oxysporum (MN633363) and F. proliferatum (MT534188), respectively, and the TEF1-α sequences of YTU1 (712 bp) and YTH1 (703 bp) shared 100% homology with F. oxysporum (MN507110) and F. proliferatum (MK952799), respectively. Sequences were deposited in GenBank with the Accession Nos. MZ157124 and MZ310443 for ITS, and MZ383814 and MZ383815 for TEF1-α. The pathogenicity of each isolate was determined on six healthy taro corms. All the taro corms were surface-disinfected with 70% alcohol and two locations from each corm were inoculated. One location was inoculated with 20 µl of conidial suspension (1×105 conidia/ml) and the other was inoculated with sterilized water as a control. All corms were incubated in a growth chamber at 25℃ and 95% relative humidity in the dark. After 15 days, all inoculated corms developed brown rot symptoms, while the non-inoculated control corms remained symptomless. The original isolates were successfully reisolated from all symptomatic corms and identified by sequencing, fulfilling Koch's postulates. F. oxysporum has been reported causing postharvest corm rot of taro in Bogor, Japan, and British Solomon Islands (Widodo et al., 2011). However, to our knowledge, this is the first report of F. oxysporum causing postharvest corm rot of taro in China and F. proliferatum causing postharvest corm rot of taro in the world. The disease poses a potential threat to taro production and should be timely assessed and properly managed.

First Report of Diaporthe phoenicicola causing leaf spot on blueberry (Vaccinium virgatum) in China.

Blueberry (Vaccinium virgatum), a member of the Ericaceae family, is an increasingly important crop in China because of its abundant nutritional benefits and economic value (Kuzmanovic et al. 2019). In October 2021, leaf spots were detected on 'Rabbiteye' blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E), which caused severe defoliation of the crop and fruit yield losses of 25% (Figure 1A). Disease surveys were conducted at that time; the results showed that disease incidence was 75.5%, observed in 151 of the 200 accessions sampled, and this disease had not been found at other cultivation fields in Nanchang. Lesions with taupe to dark brown margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions, with black pycnidia present in more mature lesions. To identify the causal agent, 10 small pieces (5 mm2) of leaf tissue excised from the lesion margins were surface sterilized in 75% ethanol solution for 30 s and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) at 25°C for 5 to 7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were floccose, dense, and white (Figure 1B.C). Black pycnidia developed on PDA at 25°C under a 12/12 h light/dark cycle for 30 days. Alpha conidia were 6.17 to 8.53 × 1.64 to 3.20 µm (average 6.94 × 2.52 µm, n = 100), aseptate, hyaline, fusiform to ellipsoidal, often biguttulate. Beta conidia were 15.26 to 25.41 × 0.92 to 1.40 µm (average 20.14 × 1.27 µm, n = 30), aseptate, hyaline, linear to hamate (Figure 1D). Based on morphological characteristics, the fungal isolates were suspected to be Diaporthe spp. (Gomes et al. 2013). To further confirm the identity of this putative pathogen, two representative isolates (LGM1 and LGM2) were selected for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), histone H3 (HIS), calmodulin (CAL), and ß-tubulin (TUB2) genes were amplified from gDNA and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), CYLH3F/H3-1b (Crous et al. 2004), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. GenBank accession numbers of isolate LGM1 and LGM2 were OM778771 to 72 for the ITS region, OM868228 to 29 for EF1-α, OM837771 to 72 for TUB2, ON206971 to 72 for CAL, ON206973 to 74 for HIS. BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences showed 99% (538/545 bp), 100% (322/322 bp), 99% (480/484 bp), 99% (459/460 bp), 99% (430/433 bp) identity, respectively, with those of Diaporthe phoenicicola (GenBank accession no. MW504735, MW514099, MW514142, MW514067, MT409304). Two maximum likelihood phylogenetic trees were built based on the sequences of ITS, EF1-α, HIS, CAL, and TUB2 by using MEGA 5. The two isolates LGM1 and LGM2 clustered with D. phoenicicola (Figure 2 and 3). The fungus was identified as D. phoenicicola by combining morphological and molecular characteristics. To evaluate the pathogenicity, three healthy young potted V. virgatum plants were spray inoculated with a conidial suspension of 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. The experiment was repeated three times, and all plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. Five days after inoculation, no symptoms were observed on control plants (Figure 1F), and all inoculated plants showed symptoms (brown flecks) similar to those observed in the field (Figure 1E). The fungus was reisolated from the infected tissues and confirmed as D. phoenicicola by morphological and molecular identification, and could not be isolated from the controls, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phoenicicola causing leaf spot on blueberry in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing specific control measures and potential sources for resistance to this disease caused by D. phoenicicola.

Endophytic bacterial community diversity in two citrus cultivars with different citrus canker disease resistance.

The selective infection of Xanthomonas citri pv. citri (Xcc) to citrus cultivars is universally known, but the relationship between endophytic bacteria and the resistance of host variety to canker disease remains unclear. In this study, endophytic bacterial populations of two citrus cultivars-the resistant satsuma mandarin and the susceptible Newhall navel orange-were analyzed through high-throughput sequencing. The results showed that endophytic bacterial community of satsuma mandarin was more abundant than that of Newhall navel orange. In addition, bacterial abundance was the highest in the spring samples, followed by that in summer and winter samples, in both the varieties. In all samples, the predominant phyla were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes; the major genera were Bacillus and Stenotrophomonas, and the main species was Bacillus subtilis. According to the analysis of the predominant bacteria in the two citrus cultivars, B. subtilis with potential antagonistic characteristics against Xcc existed universally in all samples. However, the susceptible Newhall navel oranges were abundant in Bacillus subtilis and had a relatively large number of canker-causing cooperative bacteria such as Stenotrophomonas. The results suggested that endophytic bacterial community of the two citrus cultivars had some differences based on the season or plant tissue, and these differences were mainly in the quantity of bacteria, affecting citrus canker disease occurrence. In conclusion, the differences in endophytic bacteria on citrus cultivars might be related to host resistance or susceptibility to citrus canker disease.

Transcriptome analysis of Actinidia chinensis in response to Botryosphaeria dothidea infection.

Ripe rot caused by Botryosphaeria dothidea causes extensive production losses in kiwifruit (Actinidia chinensis Planch.). Our previous study showed that kiwifruit variety "Jinyan" is resistant to B. dothidea while "Hongyang" is susceptible. For a comparative analysis of the response of these varieties to B. dothidea infection, we performed a transcriptome analysis by RNA sequencing. A total of 305.24 Gb of clean bases were generated from 36 libraries of which 175.76 Gb was from the resistant variety and 129.48 Gb from the susceptible variety. From the libraries generated, we identified 44,656 genes including 39,041 reference genes, 5,615 novel transcripts, and 13,898 differentially expressed genes (DEGs). Among these, 2,373 potentially defense-related genes linked to calcium signaling, mitogen-activated protein kinase (MAPK), cell wall modification, phytoalexin synthesis, transcription factors, pattern-recognition receptors, and pathogenesis-related proteins may regulate kiwifruit resistance to B. dothidea. DEGs involved in calcium signaling, MAPK, and cell wall modification in the resistant variety were induced at an earlier stage and at higher levels compared with the susceptible variety. Thirty DEGs involved in plant defense response were strongly induced in the resistant variety at all three time points. This study allowed the first comprehensive understanding of kiwifruit transcriptome in response to B. dothidea and may help identify key genes required for ripe rot resistance in kiwifruit.

Specific and sensitive detection of the guava fruit anthracnose pathogen (Colletotrichum gloeosporioides) by loop-mediated isothermal amplification (LAMP) assay.

Anthracnose of guava, caused by the fungus Colletotrichum gloeosporioides, is a major factor limiting worldwide guava production. Timely and accurate detection of the pathogen is important in developing a disease management strategy. Herein, a loop-mediated isothermal amplification (LAMP) assay for the specific and sensitive detection of C. gloeosporioides was developed using primers targeting the ß-tubulin 2 (TUB2) gene. The optimal reaction conditions were 64 °C for 60 min. The specificity of the method was tested against C. gloeosporioides isolates, Colletotrichum spp. isolates, and isolates of other genera. Positive results were obtained only in the presence of C. gloeosporioides, whereas no cross-reaction was observed for other species. The detection limit of the LAMP assay was 10 fg of genomic DNA in a 25 µL reaction. The LAMP assay successfully detected C. gloeosporioides in guava fruit collected in the field. The results indicate that the developed LAMP assay is a simple, cost-effective, rapid, highly sensitive, and specific tool for the diagnosis of guava anthracnose caused by C. gloeosporioides and could be useful for disease management.

Expression of dsRNA in recombinant Isaria fumosorosea strain targets the TLR7 gene in Bemisia tabaci.

BACKGROUND: RNA interference (RNAi) technology shows a great potential in controlling agricultural pests, despite the difficulty of introducing exogenous dsRNA/siRNA into target pests. Isaria fumosorosea is a common fungal pathogen of the B-biotype Bemisia tabaci (whitefly), which is a widespread pest. Entomopathogenic fungi directly penetrate the cuticle and invade insect hemocoel. Application of I. fumosorosea expressing dsRNA of whitefly immunity-related gene may aid in developing RNAi technology to effectively control whiteflies. METHODS: A dsRNA expression plasmid, psTLR7, was constructed by introducing the Toll-like receptor 7 (TLR7) gene of B-biotype whitefly to the silent vector, pSilent-1. The plasmid psTLR7 was transferred into the protoplast of the I. fumosorosea strain IfB01. Then, the recombinant strain was screened out based on the biological stability and bioactivity against whitefly. RESULTS: A genetically stable recombinant strain IfB01-TRL7 was screened out. The impact of IfB01-TRL7 against whitefly TRL7 gene was validated by qPCR. Lower expression levels of the TLR7 gene was observed in the whiteflies infected by the recombinant strain. The bioassay results indicated that compared to IfB01 strain, IfB01-TRL7 increased the mortality of whitefly nymphs, and decreased and shortened the values of LC50 and LT50, thus indicating higher virulence of IfB01-TRL7. CONCLUSION: The expression of the dsRNA of whitefly TLR7 gene in recombinant I. fumosorosea strain successfully knocked down the host target gene by infecting the nymphs and enhanced the whiteflies mortality. The present study will give insight to new application of RNAi technology for more effective biocontrol of this pests.

Genetic variability of genome segments 3 and 9 of Fiji disease virus field isolates.

Fiji leaf gall is an important disease of sugarcane in Australia and other Asia-Pacific countries. The causative agent is the reovirus Fiji disease virus (FDV). Previous reports indicate that there is variation in pathology between virus isolates. To investigate the amount of genetic variation found in FDV, 25 field isolates from Australia, Papua New Guinea and Malaysia were analysed by partial sequencing of genome segments S3 and S9. There was up to 15% divergence in the nucleotide sequence among the 25 isolates. A similar amount of divergence and pattern of relationships was found for each of the two genomic segments for most of the field isolates, although reassortment of genome segments seems likely for at least one of the Papua New Guinean isolates. The finding of a high level of variation in FDV isolated in different regions has implications for quarantine and disease management.