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Pokkah boeng disease (PBD), a sugarcane foliar disease, is caused by various Fusarium spp. within the Fusarium fujikuroi species complex (FFSC). In the current study, we investigated the diversity of Fusarium spp. associated with PBD in China. In total, 320 leaf samples displaying PBD symptoms were collected over 10 consecutive years (2012 to 2021), during winter and summer, from six various sugarcane-growing regions (Guangxi, Yunnan, Guangdong, Zhejiang, Hainan, and Fujian) in China. Phylogenetic analysis of Fusarium spp. was reconstructed using translation elongation factor 1-α, and DNA-directed RNA polymerase II largest subunit and second-largest subunit multigene sequences. Evolutionary studies of these regions categorized the isolates into four FFSC species (F. sacchari, F. proliferatum, F. verticillioides, and F. andiyazi). The identified isolates, which developed irregular necrotic patches and rotting symptoms on the sugarcane plant after approximately 30 days were tested for their pathogenicity. Symptoms that appeared during pathogenicity testing were consistent with those observed under field conditions. Each strain of the pathogenic Fusarium spp. belonged to different vegetative compatibility groups (VCGs), and there was no affinity between VCGs. Our results contribute to understanding FFSC and accurately identifying Fusarium spp. associated with the sugarcane crop.

Nano-emulsion is a promising delivery system for increasing pesticide use and enhancing the therapeutic efficiency against pathogens. The pathogen Candidatus Liberibacter asiaticus (Las) that causes destructive citrus huanglongbing (HLB) resides in citrus phloem, which makes it difficult to treat with chemicals. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, an oil-in-water (O/W) nano-emulsion formulation was developed and optimized to combat citrus HLB. The nano-emulsion was formulated through a spontaneous emulsification method for efficient delivery of ampicillin into the citrus phloem using bark application. The nano-emulsion that was prepared from Cremophor EL (viscous oil), acetone (water miscibility organic solvent), and Span 80/Tween 80 (surfactant) formed a small droplet size (17.33 ± 0.52 nm) and exhibited an improved absorption rate. Peak concentration was detected at 2 days posttreatment and the maximum concentration (Cmax) and relative bioavailability (RBA) of ampicillin in HLB-affected citrus were 71.86 ± 35.38 ng/g and 267.25% ± 44.1%, respectively. The peak concentration of Amp appeared at 6 days posttreatment in the citrus trees that were treated with Amp alone and their Cmax and RBA were 56.44 ± 32.59 ng/g and 100%, respectively. The same nano-emulsion was used to deliver five different antimicrobials to control citrus HLB through bark application. We found that the droplet size of the antimicrobials in the nano-emulsion was significantly reduced and the nano-emulsion also enhanced the therapeutic efficiency of validoxylamine A alone and in combination with actidione as well as sulfadimoethoxine sodium against Las. Therefore, this study provides an efficient bark application nano-emulsion formulation for citrus HLB control.

Sugarcane is a major sugar and the leading energy crop worldwide and Guangxi is the largest sugarcane production area in China (2011 Sugar Annual China, www.gain.fas.usda.gov). During survey of sugarcane crops in September 2012 and June 2013, ~5 to 10% of sugarcane (cvs. FN-40 and ROC22) planted in Chongzuo and Laibing, Guangxi Zhuang Autonomous Region, P. R. China, had twisted and curling symptoms of crown leaves similar to sugarcane Pokkah boeng disease (caused by Fusarium moniliforme Sheldon). The symptoms started appearing as yellowing on midribs and leaf margins that spread further to the entire leaf, along with twisting and curling of crown leaves. The symptomatic leaf tissues (5 × 5 mm) were surface-sterilized by 0.1% HgCl2 solution for 30 s, followed by rinsing three times in sterile water, placed on potato dextrose agar (PDA), and then incubated in darkness at 28°C. After 3 days of incubation, the isolated fungal colony appeared as white villous, spherical, radial, and dense colorless mycelium from the top, while it was reddish-brown at the bottom and later became grayish. Chlamydospores were also observed with a diameter of 5 to 10 μm and were dark brown, unicellular, intercalary, and smooth. The binucleate hyphae were colorless and transparent. Pycnidia appeared on the colonies after 20 days, and were dark brown, subglobose, and 150 to 230 μm in diameter, and the conidia were ~3 to 7 × 2.5 to 6 μm, unicellular, colorless, and ovoid to oval. The fungal isolates from the symptomatic leaves were obtained and pathogenicity was evaluated. Conidial suspensions (107 CFU/ml) of the single isolate from FN-40 were micro-injected into 20 sugarcane seedlings of cultivar FN-40. Another 20 seedlings were injected with water without conidia as control. The inoculated plants were grown in a growth chamber at 28°C with a 16-h photoperiod. Twisted and curly symptoms similar to the field appeared on the inoculated leaves at 10 days after inoculation, while the control leaves remained asymptomatic. The fungus was re-isolated and identified. Genomic DNA from the cultured fungal isolate was extracted with a modified Fungal DNA Midi Kit (Omega Bio-Tek, Inc., Norcross, GA), and amplified using fungus-conserved primer sequences (ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ and ITS4: 5′-TCCTCCGCTTATTGATATGC-3′). The consensus rDNA-internal transcribed spacer sequence (GenBank Accession No. KC524502) was 100% identical with 97% coverage to the ITS sequence from Phoma sp. 3. TMS-2011 (HQ631000.1) in GenBank (3). The fungus Phoma sp. was identified on the basis of morphological characteristics (2,4) and the ITS sequence of rDNA (1,3). Disease caused by Phoma sp. has been reported earlier on sugarcane from Pakistan, Hawaii, and Taiwan, causing leaf blight and curling (2,4). However, to the best of our knowledge, this is the first report of Phoma sp. causing twisting and curling of crown leaves of sugarcane in mainland of China. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) A. Sanguino and H. Tokeski. ISSCT Proc. 17:1555, 1980. (3) P. Shrestha et al. Appl. Environ. Microbiol. 77:5490, 2011. (4) Z. N. Wang. Rep. Taiwan Sugar Res. Inst. 129:1, 1980.

Sugarcane twisted leaf disease, caused by Phoma sp., was first reported in Guangxi, China, in 2012, when more than 5% of sugarcane was infected in the field. Three single-spore isolates were recovered from symptomatic leaves. Sequences from five fungal loci, 28S nrDNA (LSU), 18S nrDNA (SSU), the internal transcribed spacer regions 1 and 2 and 5.8S nrDNA (ITS), β-tubulin (TUB), and the translation elongation factor alpha (TEF-α) were amplified from the disease-associated isolates. The twisted leaf disease pathogen was identified and formally described as Phoma sorghina var. saccharum through phylogenetic analyses, morphological observations, and the pathogenicity of the isolates on sugarcane. P. sorghina var. saccharum can be differentiated from related species based on the morphology of pycnidia and chlamydospores that formed regular, glabrous, papillate ostioles. Chlamydospore-anamorph was unicellular, botryoid-alternarioid shape, as well as the binucleate, frequently branched hyphae. We also showed that mycelial growth of P. sorghina var. saccharum was optimal at pH 4.0 and 20 to 25°C. Additionally, among 13 chemical compounds tested, carbendazim was found to be the most effective in suppressing the radial growth of the fungus. Mycelial growth in vitro was completely inhibited at concentrations of 100 and 50 ppm, and 87.6% of mycelial growth was inhibited at 10 ppm. Carbendazim is therefore a potentially effective fungicide to control this disease in China.

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria-Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 μl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

China is the world’s third-largest sugar producer after India and Brazil, with sugarcane plants accounting for 90% of total sugar production in China. Susceptible sugarcane cultivars were vulnerable to severe damage by wilt, which in China has caused yield losses of about 10.0%. During a disease survey in a nationwide test of sugarcane varieties in different regions in August 2019, 5-month-old plants of the sugarcane hybrid variety Zhongzhe1 (ZZ1) grown in Beihai (109°06′52″E, 21°28′37″N) of Guangxi Zhuang Autonomous Region were found to exhibit dried internodal tissue, yellowed leaves, and hollow stalks consistent with wilt. A total of 18 sugarcane stalk samples affected by symptomatic wilt were collected from Beihai. Symptomatic internal stalk tissues were dissected into 0.5 × 0.5-cm pieces that were surface sterilized with 0.1% HgCl₂ for 45 s, followed by 70% ethanol for 60 s, and rinsed three times with sterile water. Around 5 to 10 pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for 2 to 3 days. Growing hyphal tips were picked and transferred to fresh PDA plates to purify fungal isolates and maintained on PDA and carnation leaf-piece agar (CLA) for observing conidia and conidial fructification. A total of six fungal strains were isolated from the 18 symptomatic stalk samples (Ho and Ko 1997). These isolates produced abundant white mycelia on the PDA medium for 3 days. The macroconidia typically had three to five septa and were slightly falcate, relatively slender, and had thin walls. The microconidia were slim, oval, and primarily nonseptate. The microconidia grown on CLA had an oval shape that was similar to Fusarium sacchari (Leslie and Summerell 2006), which had no long chains and produced false heads from mono- and polyphialides. Genomic DNA from the six single-spore cultured isolates was extracted, amplified, and sequenced using two specific primer pairs of the translation elongation factor TEF1-α (EF1/EF2, 0.6 kb) and the second-largest subunit of RNA polymerase II, RPB2 (5f2/7cr and 7cf/11ar, 1.8 kb) (O’Donnell et al. 2013). All 12 sequences were submitted to GenBank (MN894584 to MN894595) and had over 99% homology to F. sacchari with 657/657 for TEF1-α (MK152503) and 1,700/1,706 for RPB2 (MK829741) through pairwise alignment against the Fusarium MLST database. A phylogenetic tree on the TEF1-α and RPB2 gene sequences was constructed by neighbor-joining methods with the reference sequences from other Fusarium species, including F. sacchari, F. mangiferae, F. fujikuroi, F. proliferatum, F. thapsinum, and F. verticillioides. Sequence alignment confirmed that the six isolates belonged to F. sacchari. A pathogenicity test was conducted on 5-month-old cultivar ZZ1. Stalk inoculation was achieved through a borehole made at the third internode from the top visible dewlap of the plant using a 5-mm-diameter hole punch (Viswanathan et al. 2011). A mycelial plug of the three strains isolated from wilt-affected plants (GX-BH01, GX-BH02, and GX-BH03) was placed in the borehole and sealed with Parafilm. A plug of the sterile medium was used as a control. All pathogenicity assays were repeated three times. The inoculated plants were placed in a growth chamber at 25 to 28°C with a 12-h day/12-h night photoperiod. After 30 days, the canes inoculated with the fungal isolates exhibited tissue discoloration and hollow stalks that were similar to those observed in field-grown sugarcane, whereas the control plants had no symptoms. The reisolated fungi were identified as F. sacchari based on morphological, cultural, and molecular characteristics. Our results confirmed that F. sacchari is the causative pathogen of sugarcane wilt, and this is the first report of this pathogen in China. This report has epidemiological significance for the planning of suitable management practices to prevent wilt disease in China.

Anubias barteri var. nana (belonging to family Araceae) is one of the most popular ornamental plants for aquaria. It has slow growth and small attractive ornamental leaves. It originated from West Africa, was first described in 1860 by Heinrich Wilhelm Schott (1), and is now commercially planted widely in southern China. A new disease of A. barteri var. nana was observed on leaves. Approximately 30% of the plants in an ornamental nursery in Baiyun district, Gaungzhou, China (113°22′45.15″ E, 23°23′41.15″ N) in July 2013 were found to be infected. The pathogen caused necrotic spots that progressed to form holes on leaves of the infected plants, negatively affecting their ornamental market value. Eventually the diseased plant died. White fungal fruiting bodies and black sporodochia were observed on the surfaces of the symptomatic leaves. A single-spore isolate (myr5) was obtained from the concentric lesions and cultured on potato dextrose agar (PDA). The floccose fungal colonies were white to buff, and black conidiomata were often visible on the surface after 25 days of incubation. The conidia had rounded ends and the average size of conidia was 5.98 ± 0.15 × 2.24 ± 0.08 μm. The rDNA internal transcribed spacer (ITS) region of isolate myr5 was amplified by PCR using ITS1 and ITS4 primer pairs. The amplified product was sequenced and deposited in GenBank (Accession No. KJ572115) and showed 99% identity to Myrothecium roridum isolates BBA 71015 (AJ302001) and BBA 67679 (AJ301995) (unpublished), and 100% identity to the isolate myr2-2 from Dieffenbachia picta ‘Camilla’ in Taiwan (2). On the basis of morphological and molecular characterization, the fungus causing leaf spot on A. barteri var. nana plants was identified as M. roridum Tode ex Fr (2,3). To confirm the etiology of the disease, Koch's postulates was performed in a greenhouse (28 ± 2°C) using 3-day-old cultures of isolate myr5 and fungal spore suspensions of 1 × 105 conidia ml−1 containing 0.05% of Tween 20. Sixteen healthy leaves, two each from eight plants, were infiltrated on two different parts close to the midribs with 100 μl of the fungal spore using needleless syringes. Water infiltration was used as the control treatment. Water-soaked brown lesions appeared on leaves 7 days after inoculation, followed by the development of dark concentric rings within the necrotic areas on the surface of the inoculated leaves after 15 days of incubation. These symptoms were similar to those in the naturally infected aquarium plants. No symptoms were observed on any of the water micro-infiltrated plants. The fungus was re-isolated from the inoculated plants, but not from control plants. To our knowledge, this is the first report of Myrothecium leaf spot caused by M. roridum on A. barteri var. nana in mainland China. References: (1) W. Crusio. Primitiae Africanae XII 79 (14):1-48, 1979. (2) C. F. Hong et al. Plant Dis. 97:1253, 2013. (3) M. Tulloch. Mycol. Pap. 130:1-42, 1972.