EF1-α Gene Sequences Research Articles

First Report of Fusarium Stem and Root Rot of Gerbera jamesonii Caused by Fusarium incarnatum in China.

Gerbera (Gerbera jamesonii Bolus) is an important cut flower grown globally. In 2020, gerbera plants (Redaicaoyuan, Baimawangzi, and Hongditan cultivars) with roots, crowns, and stems rot were found in a greenhouse in Nanping, Fujian, China. Approximately 30% of the 60,000 plants showed symptoms. Diseased plants were stunted with chlorotic leaves. The leaves and flower heads were wilted and withered. Brown discoloration with red to black streaks occurred in the vascular system of the crown and stem. The stem pieces (3×3 mm) showing the symptom were surface-disinfected with 1% NaClO for 1 min and washed three times with sterilized water. The stem pieces were then dried and placed on potato dextrose agar (PDA) at 25℃ inside a dark chamber. Ten single-spored isolates were identified asFusarium incarnatumbased on morphological features. White to light brown mycelia were observed among the isolates on PDA medium. Falculate, multicelluar, straight to slightly curved macroconidia produced in monophialide sporodochia without distinctive foot shaped basal cell; and chlamydospores produced in some isolates (Leslie and Summerell). The size of macroconidia was 36.4 ± 5.20 × 4.6 ± 1.3 μm (n = 100) with 3 to 5 septates. Microconidia were mostly 0 to 1 septate measured 14.6 ± 1.9 × 2.6 ± 0.5 μm (n=100). Based on the morphological observation, isolates were further identified by molecular method. The ITS1/4 region combined with partial gene fragments of translation elongation factor (EF-1α, primer EF1/EF2, Geiser et al.) and calmodulin (CAM, primer CL1/CL2A, O'Donnell.) from the isolates were amplified and sequenced. All of the three tested isolates showed identical gene sequences. Sequences amplified from one represented isolate FIN-1 were submitted to Genbank. BLAST searches revealed that ITS1/4 (MW527088), EF-1α (MW556488), and CAM (MW556487) had 99.22%, 99.53%, 99.42% identity compared to F. incarnatum (MN480497, MN233577, and LN901596, respectively) in GenBank. FUSARIUM-ID (Geiser et al. 2004) analysis also showed 99 to 100% similarity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01636 for CAM, FD_01643 for EF-1α). The phylogenetic analysis was conducted using neighbor-joining algorithm based on the ITS, EF-1α, and CAM gene sequences. The isolate was clustered with F. incarnatum clade. Then, the pathogenicity of the fungus was confirmed by performing Koch's postulates. Pure single-spored cultures were grown on carboxymethyl-cellulose (CMC) medium for sporulation. G. jamesonii plants used for pathogenicity tests were grown on sterilized potting soil in a plastic container to the ten-leaf stage prior to inoculation. Spores harvested from the CMC medium were adjusted to a concentration of 1×105 conidial/ml. Twelve healthy rooted gerbera seedlings were inoculated by drenching 10 ml of the conidial suspension onto roots. Twelve gerbera seedlings treated with 10 ml sterile water served as control treatments. Plants were grown in the glasshouse at temperatures of 23°C, relative humidity >70%, and 16 h light per day. After 10 days, blackening stems and withered leaf edges began to appear on inoculated seedlings, whereas control seedlings remained healthy. F. incarnatum was consistently re-isolated from the symptomatic stems, whereas no isolates were obtained from the control seedlings. The assay was conducted twice. To the best of our knowledge, this is the first report of F. incarnatum causing stem and root rot on G. jamesonii.

First Report of Fusarium Root Rot of Tobacco Caused by Fusarium sinensis in Henan Province China.

Tobacco (Nicotiana tabacum L.) is an economically important crop in China, with an estimated production of 2.2 million tons every year. In June 2018, tobacco plants within the municipality of Sanmenxia (Henan, China) showed symptoms of wilting with leaf yellowing and stunting. Diseased plants exhibited severe necrosis that advanced through the main root (Figure 1 A). The symptoms were observed in nineteen surveyed tobacco fields, 60 ha in total, and approximately 25% of the plants were symptomatic. The disease resulted in a severe loss in tobacco leaf production. Five symptomatic tobacco plants were sampled. Diseased tissues from roots were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Eighteen of the 25 diseased tissues had cultures growing from them, and all the cultures were white colonies with abundant aerial mycelium produced scarlet pigmentation on PDA. One pure culture was obtained by single-spore culturing (SL1). A 10-day-old culture grown on CLA (carnation leaf agar) produced macroconidia that were falcate, straight or slightly curved, 3-septate, 25-35×3.5-4.5 μm (average 26.8×3.7 μm) (n=50). Two types of microconidia (napiform and fusiform) were formed on CLA that were hyaline, with one to two cells. Napiform conidia were 4.5-9.3×3.8-5.9 (average 7.3×5.0 μm) (n=50); fusiform conidia were 6.9-15.8×1.8-3.1 (average 9.9×2.5 μm). Spherical chlamydospores (7-12.5 μm) (n=50) were terminal or intercalary and produced in clumps or in chains (Figure1 B-D). Morphological characteristics of the isolate were similar to the features of Fusarium sinensis previously described by Zhao and Lu (2008). Molecular identification was performed using partial sequences of EF1-α gene (primers EF1/EF2, O'Donnell et al. 1998). Maximum parsimony and maximum likelihood-based methods were fitted using MEGA 7 (Moreira et al. 2019，Figure 2). The isolate was also sequenced for β-tubulin (primers T1/Bt-2b, O'Donnell & Cigelnik 1997)，ribosomal RNA gene (LSU, LROR/LR5 primers, Vu et al. 2019) and rDNA-ITS (ITS 1/ ITS 4 primers, White et al. 1990). Sequences were deposited in GenBank under accession numbers MT947797 (EF1-α), MW484999 (β-tubulin), MW486649 (LSU) and MT907471 (ITS). The obtained EF1-α sequence was 98.10% identity with those of F. sinensis (MG670388.1) in the GenBank database, whereas the β-tubulin, LSU and ITS sequences showed 100% identities to the corresponding DNA sequences in F. sinensis (GenBank Acc. Nos. KX880370.1, NG_067454.1 and MH863232.1, respectively). Morphological and molecular results confirmed this species as F. sinensis (Zhao and Lu 2008). Pathogenicity tests were performed on tobacco seedlings grown on an autoclaved matrix (YC/T310-2009). Healthy 6-leaf stage tobacco seedlings were inoculated by pouring a 20 mL conidial suspension (1×106 conidia/mL-1) around the stem base of each plant, 30 plant were inoculated. Thirty control seedlings received sterilized water. All treatments were maintained for 30 days under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Root rot and foliage chlorosis similar to the ones observed on infected plants in the field were observed on the inoculated tobacco seedlings, whereas the control seedlings remained asymptomatic after 30 days (Figure1 E). The pathogen isolated from the inoculated plant exhibited morphological characteristics identical to F. sinensis and was identified by a partial EF1-α gene sequence. This disease has previously been reported as the causal agent of root and crown rot of wheat in China (Zhao and Lu 2008; Xu et al. 2018). To our knowledge, this is the first report of F. sinensis causing root rot on tobacco in China. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010).

Phylogeography of the endemic red-tailed cicadas of New Zealand (Hemiptera: Cicadidae: Rhodopsalta), and molecular, morphological and bioacoustical confirmation of the existence of Hudson’s Rhodopsalta microdora

Abstract Why do some genera radiate, whereas others do not? The genetic structure of present-day populations can provide clues for developing hypotheses. In New Zealand, three Cicadidae genera are depauperate [Amphipsalta (three species), Notopsalta (one species) and Rhodopsalta (three species)], whereas two have speciated extensively [Kikihia (~30 species/subspecies) and Maoricicada (~20 species/subspecies). Here, we examine the evolution of Rhodopsalta, the last New Zealand genus to be studied phylogenetically and phylogeographically. We use Bayesian and maximum-likelihood analyses of mitochondrial cox1 and nuclear EF1α gene sequences. Concatenated and single-gene phylogenies for 70 specimens (58 localities) support its monophyly and three described species: Rhodopsalta cruentata, Rhodopsalta leptomera and Rhodopsalta microdora, the last taxon previously regarded as uncertain. We provide distribution maps, biological notes and the first descriptions of diagnostic songs. We show that both R. cruentata and R. microdora exhibit northern and southern genetic subclades. Subclades of the dry-adapted R. microdora clade show geographical structure, whereas those of the mesic R. cruentata and sand-dune specialist R. leptomera have few discernible patterns. Genetic, bioacoustical and detailed distributional evidence for R. microdora add to the known biodiversity of New Zealand. We designate a lectotype for Tettigonia cruentataFabricius, 1775, the type species of Rhodopsalta.

Warts Galore – on Three New Lamprospora De Not. Species (Pezizales) from Southern Europe and Macaronesia and a Type Revision of Three Species Described from the US by F. J. Seaver in the 1910s

Lamprospora bulbiformis M.Vega & Janošík, sp. nov., L. gibbosa M.Vega & Janošík, sp. nov. and L. thelespora Martínez-Gil, M.Vega & E.Rubio, sp. nov. are described and illustrated based on live collections from Cyprus, France, Portugal and Spain. Phylogenetic analyses of the concatenated LSU, SSU and EF1-α gene sequences show the studied collections of the three species form well supported monophyletic clades. Lamprospora bulbiformis sp. nov. infects Fissidens viridulus (Sw. ex anon.) Wahlenb., L. gibbosa sp. nov. has F. crassipes Wilson ex Bruch & Schimp. as host and L. thelespora sp. nov. grows on Cheilothela chloropus (Brid.) Broth. Three Lamprospora De Not. species namely L. tuberculata Seaver, L. tuberculatella Seaver and L. spinulosa Seaver with a slightly similar ascospore ornamentation were described by Seaver from US collections. Results of our studies of their types and additional material collected by Seaver are presented. The host of L. tuberculata is Pleuridium subulatum (Hedw.) Rabenh., that of L. tuberculatella is a species of Weissia Hedw. and that of L. spinulosa is Physcomitrium pyriforme (Hedw.) Bruch & Schimp. It has yet to be proven that species of Lamprospora described from North America also occur in Europe. European collections assigned to any Lamprospora described from North America require revision. It is not unlikely that many or even all of them represent taxa yet to be described. A considerable part of existing literature on bryophilous Pezizales needs to be reevaluated.

First Report of Dieback Caused by Neofusicoccum batangarum in Cashew in Guinea-Bissau.

Cashew (Anacardium occidentale L.) is a cash crop with a highly significant economic importance in West Africa, particularly in Guinea-Bissau (Monteiro et al. 2015, 2017). In October 2018, dieback-like symptoms such as wilt and necrosis of apical shoots were observed in 10 % of the cashew trees grown in a 100 plant-orchard in Bolama Island at Bijagós archipelago, Guinea-Bissau. Six symptomatic apical shoots from individual plants were collected for fungal isolation and identification. Tissue pieces (3 × 2 mm) from healthy to diseased margins were surface sterilized with 1 % sodium hypochlorite, washed twice with sterilized water, placed on potato dextrose agar (PDA, Difco® Laboratories) supplemented with potassium thiocyanate (50 µg/ml), and incubated at 24 ± 1 °C in the dark for 7 days. Four fungal colonies were isolated (67 %) and purified through hyphal tips removal, displaying rapid growth rate, and aerial mycelia that initially was white, turning later to dark greenish on PDA. Pycnidia produced on 1.5 % water agar and sterilized pine needles (± 25ºC; near-UV light) were solitary, covered by mycelium, obpyriform to ampulliform (152.5 ± 41.6 × 135.2 ± 30.8 μm, n = 30). Conidia were unicellular, hyaline, smooth, fusoid to ovoid, thin-walled, measuring 16.21 ± 1.52 × 5.84 ± 0.66 μm (n = 50, L/W 2.8). Such morphological features are characteristic of Neofusicoccum spp. (Phillips et al. 2013). For molecular identification, genomic DNA was extracted from a representative isolate GB160 and partial regions of ribosomal internal transcriber spacer (ITS) (ITS1/ITS4; White et al. 1990), translation elongation factor 1-α (EF1-α) (EF1-688F/EF1-1251R; Alves et al. 2008) and β-tubulin (β-tub) (Bt2a/Bt2b; Glass and Donaldson 1995) genes were amplified as previously described, respectively, with BSA (50 mg/ml). Amplicons were sequenced and deposited in GenBank (ITS, MN952993; EF1-α, MN952204; β-tub, MN952208). BLAST analysis of ITS, EF1-α and β-tub gene sequences showed 100 % identity with Neofusicoccum batangarum reference strain CBS124923 (FJ900608, FJ900654, FJ900635, respectively). Maximum-likelihood and Bayesian Inference analyses from the concatenated dataset placed GB160 isolate within the N. batangarum cluster (BS = 72 %; PP = 0.95). For pathogenicity assessment, 3-month-old cashew "Caju di Terra" plants (n = 8) grown in a greenhouse under controlled conditions were inoculated following a randomized block design as described by Lima et al. (2013). Briefly, 3 mm diam. stem tissue bark was removed and replaced with a 3 mm diameter PDA plug retrieved from the colony margin. Inoculation wound was covered with sterilized wet cotton and sealed with parafilm. Eight control plants were only treated with PDA plugs and the wound covered and sealed as described. After 15 days, all inoculated plants displayed similar symptoms to those observed in the field, and vascular lesions (10.8 ± 4.0 cm), whereas control plants remained symptomless. Koch's postulates were fulfilled by successful re-isolation of the pathogen from all inoculated stems and identification by morphology and gene sequencing. N. batangarum was identified associated with Anacardium spp. in Brazil (Netto et al. 2017) and recently reported as causing grapevine dieback in Brazil (Rêgo et al. 2020). To our knowledge, this is the first report of N. batangarum causing cashew dieback in Guinea-Bissau and West Africa. Occurrence of this disease may represent a significant impact for cashew production since this crop is the major agricultural commodity in Guinea-Bissau.

First Report of Fusarium wilt of Coleus forskohlii Caused by Fusarium oxysporum in China.

Coleus forskohlii (Wild) Briq. is an aromatic plant in the Lamiaceae family cultivated primarily in India, Sri Lanka, Nepal and China (Yunnan Province). This herb is considered to have medicinal properties and the whole plant can be used to treat asthma, cancer and other diseases with remarkable efficacy. Due to the high medicinal and economic value of C. forskohlii, it has been introduced to Tongcheng (N29°18'12.24″, E113°53'59.36″), Hubei Province for cultivation. However, severe Fusarium wilt disease of C. forskohlii has been epidemic in Tongcheng since 2018 with a disease incidence of 5 to 30% in surveyed fields. This disease is characterized typically by root rot, vascular discoloration and leaf wilting of C. forskohlii (Fig 1), resulting in progressive plant death. Ten diseased plants were collected from the fields and the roots and stems were rinsed in 70% ethanol for 5 min and samples at the junction of disease and healthy tissues (0.5 × 0.5 cm2) were cutted and placed on potato dextrose agar (PDA) for fungal isolation in a dark chamber at 28°C. Eventually, ten pure isolates were obtained from hyphal-tip followed by single-spore purification on PDA. Seven of the purified isolates showed white aerial mycelium initially and secreted orange-brown pigment 8 days after incubation. Macroconidia were falciform, hyaline, three to five septate, ranging from 2.02 to 4.17 (mean 2.98 µm) × 10.05 to 21.90 µm (mean 12.04 µm) in size (n = 30) (Fig 2). These morphological characteristics resembled Fusarium oxysporum. (Leslie and Summerell 2006) and we selected one of them for molecular identification. Genome DNA was extracted from isolate (RS-4) using the CTAB method (Mahadevakumar et al. 2018). The translation elongation factor 1 alpha (EF-1α) DNA sequence was amplified using primers EF1/EF2 (Geiser et al. 2004), and the second largest subunit of RNA polymerase II (RPB2) DNA sequence was amplified using primers fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained EF-1α sequence of RS-4 (MW219142) showed 100% identity with that of F. oxysporum (FD_01376) (FUSARIUM-ID database). RPB2 sequences of RS-4 (MW219143) showed 100% identity with F. oxysporum (FD_01679) (FUSARIUM-ID database). Moreover, a phylogenetic tree of the EF-1α gene sequence of RS-4 was constructed based on the Neighbor-Joining method in MEGA7 software (Tamura et al. 2013) and revealed that strain RS-4 was closest to F. oxysporum (Fig 2). To test the pathogenicity of RS-4, six healthy leaves of C. forskohlii were collected and inoculated either with the colonized PDA discs (diameter, 5 mm) of RS-4 or control PDA discs, in a moist chamber at 25 ± 2°C. Five days later, brown-black lesions were observed on all inoculated leaves. However, the non-inoculated leaves were maintained asymptomatic. For in vivo pathogenicity test, twenty-day-old C. forskohlii plants (n=3) were inoculated with 106 spores/ml of the RS-4 at a position approximately 1 cm above the soil. Three seedlings treated with sterile water were used as controls. These inoculated and control seedlings were incubated in a moist chamber (25 ± 2 °C, RH 85%). Three days later, typical Fusarium rot symptoms were observed on all inoculated seedlings with rotten stems and withering leaves (Fig 2). Fungal pathogens were re-isolated from the inoculated sites of in vitro and in vivo inoculations by repeating the above isolating operation, and were reconfirmed through morphological features. This is the first report of F. oxysporum causing root rot on C. forskohlii in China. F. oxysporum is one of the most economically important fungal pathogens causing vascular wilt on a wide range of plants worldwide (Dean et al. 2012). The identification of F. oxysporum as the causal agent of the observed Fusarium wilt on C. forskohlii, is critical to the prevention and control of this disease in the future. Acknowledgement This research was supported by funding from the Key project at the central government level titled, "The ability to establish sustainable uses for valuable Chinese medicinale resources" (2060302) Reference Dean, R., et al. 2012. Mol. Plant. Pathol. 13: 414. https://doi.org/10.1111/j.1364-3703.2011.00783.x. Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110: 473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0. Leslie, J. F. and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16: 1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Mahadevakumar, S. et al. 2018. Eur. J. Plant Pathol. 151:1081. https://doi.org/10.1007/s10658-017-1415-2. Tamura, K., et al. 2013. Mol. Biol. Evol. 30: 2725. https://doi.org/10.1093/molbev/msw054.

First report of Leaf Wilt caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia.

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in 'Deglet Nour' trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O'Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned 'Deglet Nour' plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch's postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 μm (avg. 13.8 μm) × 4.1 to 6.7 μm (avg. 5.1 μm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

Survey for toxigenicFusarium species on maize kernels in China

Maize is currently the most important crop in China. A major concern in maize production is maize ear rot caused byFusarium spp., which results in yield losses, reduction of seed quality and the accumulation of mycotoxins in the harvested grains. To identify the importance of the differentFusarium species in maize infection, we performed a comprehensive survey on 9,000 asymptomatic and randomly collected maize kernels. Seeds were collected from 12 different provinces covering all major maize growing areas in China and included five maize varieties. In total 1,022Fusarium isolates were retrieved that were identified based on morphological characteristics, by species specific diagnostic PCRs and by EF1-α gene sequencing. Eight different species were identified:Fusarium verticillioides (75.34%),Fusarium graminearum (8.32%),Fusarium proliferatum (7.14%),Fusarium subglutinans (4.11%),Fusarium meridionale (1.57%),Fusarium oxysporum (1.37%),Fusarium semitectum (1.17%), andFusarium asiaticum (0.98%). The distribution ofFusarium species was found to be different in different regions with the largest diversity observed in Hubei province, where all eightFusarium species were isolated. Genetic chemotyping within theF. graminearum species complex indicated that all of the 85F. graminearum isolates showed the 15-acetyldeoxynivalenol chemotype, whereas allF. asiaticum (n=10) andF. meridionale (n=16) isolates had the nivalenol chemotype even when isolated from the same maize field. To our knowledge this is the largest collection ofFusarium isolates from maize and further exploitations of this collection are discussed.

Occurrence of Southern Blight Caused by Athelia rolfsii on Pinellia ternata in China and Worldwide

Pinellia ternata (Thunb.) Breit. is an herbaceous plant (family Araceae) native to China. The tuber of P. ternata is used in traditional Chinese herbal medicine as an antiemetic and antitussive (Marki et al. 1987). In June 2018, a soilborne disease on P. ternata plants was initially observed in Zhongxiang City (31°17′N, 112°58′E), central Hubei Province, China. The incidence of the disease was 25 to 30% in two commercial fields, and the total area affected by the disease was approximately 200 ha. Symptomatic plants initially had small, water-soaked, sunken lesions on the stems near the soil line that developed symptoms typical of southern blight. As the disease progressed, white hyphae were observed on infected stem lesions in the presence of abundant moisture. Numerous tan-to-brown, spherical sclerotia developed on the infected basal stem and on soil surfaces around the infected plants. After 1 month, plant foliage became wilted, followed by a complete collapse of the plant. Sclerotia from symptomatic basal stems of plants were surface disinfested in 2% sodium hypochlorite for 1 min, washed thrice with sterilized distilled water, air dried, and placed on potato dextrose agar (PDA). The cultures were incubated at 28 ± 1°C in the dark. Numerous globoid sclerotia formed on PDA after 12 days of growth. The sclerotia, 2 to 4 mm in diameter, were white at first and became melanized, turning brown with age. The white mycelium formed typical clamp connections after 5 days of growth. Aerial fungal growth was composed of many narrow hyphae 3 to 8 μm wide. The isolate was identified as Athelia rolfsii on the basis of mycelial and sclerotial characteristics (Punja and Damiani 1996). To confirm the identification, the strain NB1 was chosen for DNA sequencing. The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primers (White et al. 1990) and sequenced, and the resulting sequences of 650 bp were deposited in GenBank (accession no. MN071107). BLAST analysis of the sequences showed 99.8% identity to A. rolfsii (imperfect stage of Sclerotium rolfsii) (MN328049.1). Moreover, the isolate was further identified to the species level as A. rolfsii by amplifying RPB2 gene sequences (accession no. MN509436) and EF1-α gene sequences (accession no. MN509438), which also exhibited 100% similarities with A. rolfsii (JF267830 and JF267817) (Remesal et al. 2013). Based on morphological and molecular characteristics, the fungus was identified as A. rolfsii. NB1 was chosen to fulfill Koch’s postulates; 10 healthy 30-day-old P. ternata seedlings were planted in plastic pots with sterilized mixture of peat moss and vermiculite (3:1). Each seeding was inoculated with one sclerotium, which was placed on the soil surface against the stem of the plant. Three noninoculated plants served as controls. The inoculated and noninoculated plants then were placed in a greenhouse at 28 ± 1°C, with ambient lighting and relative humidity of 70%. By 10 days after inoculation, all inoculated plants showed symptoms identical to those observed in the field, and no symptoms were observed on control plants. The fungus was reisolated from the inoculated plants and identified by the above method. The experiment was repeated thrice with similar results. To our knowledge, this is the first report of southern blight caused by A. rolfsii on P. ternata in China and worldwide. There is a need to develop effective management measures to reduce the occurrence of this disease.

First report of root rot caused by Fusarium oxysporum on Macleaya cordata in China

Macleaya cordata is a perennial herb that belongs to the family Papaveraceae and, as a commercial crop, is widely used in the livestock industry. Root rot disease is one of the most serious diseases of M. cordata. In June 2017, a brown root rot was observed on M. cordata, cv. HNXN-001 in 10 commercial fields in Hunan Province, China. The fields varied from 1000 m2 to 3500 m2, with approximately 8% of plants affected. Symptoms included withering, leaf chlorosis, brown rot of the root tips or lateral roots, and internal decay of the taproots leading to plant death. Fusarium oxysporum Schlecht. was isolated and identified on the basis of morphology and ITS rDNA and EF-1α gene sequencing. This is the first report of root rot of M. cordata caused by F. oxysporum in China. Detection of F. oxysporum on diseased M. cordata roots is important for M. cordata root rot control.

Sequencing of cytochrome c oxidase subunit I (COI) gene and elongation factor 1-alpha (EF1α) gene in aphids of the Belarussian fauna

Aim. Cytochrome c oxidase subunit I gene (COI) and elongation factor 1-alpha gene (EF1α) are frequently used for a correct species diagnostics of aphids forms that are correct diagnosis of the species, studying of species genetic structure, assessment of intraspecies and interspecies genetic polymorphism assessment and construction of phylogenetic systems. Methods. Aphids samples were collected in Belarus. Genetic sequences of COI and EF1α genes were sequenced using primes LCO and EF3. Results. Sequences of COI and EF1α genes of 18 aphid species of Belarussian fauna including 6 aphid species of COI gene (Aphis fabae mordvilkoi Börn. & Janisch,, Aphis pomi Deg., Aphis spiraecola Patch, Colopha compressa Koch, Panaphis juglandis (Gz.) и Uroleucon hypochoeridis (F.)) and 15 species of EF1α gene (Anoecia corni (Fabr.), Aphis euphorbiae Kalt., C. compressa, Drepanosiphum platanoidis (Schr.), Gyphina jacutensis Mordv., Hyalopterus pruni (Geoffr.), Longicaudus trirhodus (Walk.), Monaphis antennata (Kalt.), P. juglandis, Periphyllus aceris (L.), Schizolachmus pineti (F.), Sipha maydis Pass., Therioaphis tenera Aiz., Trichosiphonaphis corticis (Aiz.), U. hypochoeridis) were obtained. Conclusions. COI gene and EF1α gene sequences were decoded and deposited to GenBank.
 Keywords: aphids, COI, EF1α, nucleotide sequence, GenBank, BOLD.

The Construction of Full-Length cDNA Library for Otodectes cynotis.

Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/μl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.

Relationships among Brazilian and worldwide isolates of Fusarium oxysporum f. sp. lactucae race 1 inferred from ribosomal intergenic spacer (IGS-rDNA) region and EF-1α gene sequences

Fusarium wilt, caused by Fusarium oxysporum f. sp. lactucae (FOLac), is amongst the main diseases affecting lettuce in subtropical regions. Although nationwide surveys indicated the exclusive presence of FOLac race 1 in Brazil, no detailed studies are available providing molecular evidences if these isolates were introduced into the country via contaminated seeds or if they are endemic populations. The translation elongation factor 1α (EF-1α) gene and rDNA intergenic spacer (IGS-rDNA) region represent the most comprehensive databases for comparative analyses of Fusarium isolates. Our aim was to assess the genetic relationships of 23 Brazilian FOLac race 1 isolates with a collection of FOLac isolates of worldwide origin, using the information from these genomic regions. A consistent single-cluster pattern was observed for FOLac race 1 isolates from Brazil, California-USA, Arizona-USA, Japan, Italy, as well as the novel FOLac race 4 isolates from the Netherlands based upon the EF-1α (604 nucleotides) and the IGS-rDNA (1859 nucleotides) sequences. Our analysis (based upon six single nucleotide polymorphisms identified only in the IGS-rDNA sequence) allowed the identification of intra-race 1 variation with the discrimination of four haplotypes. Isolates from Brazil, Italy, and a subset from the USA were classified into a single haplotype. The low diversity levels and the presence of only a single haplotype across the entire country are strong indications that Brazilian FOLac race 1 isolates are result of recent introduction event(s). This fast and widespread distribution of FOLac race 1 in Brazil has occurred more likely via importation and planting of contaminated seeds.

CARACTERIZACIÓN MOLECULAR Y PATOGÉNESIS DE Fusarium ASOCIADO AL AMARILLAMIENTO DEL TRIGO

El amarillamiento o madurez prematura del trigo (Triticum aestivum L.) es una enfermedad causada por un complejo de especies del género Fusarium que ataca cualquier parte de la planta. Este problema se ha reportado en diversos municipios del estado de Guanajuato, México desde el año 2000. El objetivo del presente trabajo fue aislar, caracterizar e identificar especies de Fusarium asociadas al amarillamiento del cultivo de trigo en ocho localidades de Guanajuato y cuantificar su patogenicidad. Se obtuvieron 116 aislamientos de diferentes órganos de plantas con síntomas que fueron caracterizados molecularmente con ITS-RFLP, ISSR e identificados con las secuencias del gen EF1-α. Los fragmentos RFLP de la región ITS y la técnica ISSR conformaron distintos agrupamientos. Las secuencias del gen EF1-α identificaron cercanamente a los aislamientos con las especies F. proliferatum, F. subglutinans, F. oxysporum, F. thapsinum y F. andiyazi. Se observó gran amplitud de severidad en las pruebas de patogenicidad (de 2.2 a 39.7 %). La mayor agresividad se identificó en la cepa T5.10 (F. proliferatum), así como en efecto conjunto de las cepas R8.5 (F. proliferatum) y E5.3 (no identificada).

Ecological preferences of Metarhizium spp. from Russia and neighboring territories and their activity against Colorado potato beetle larvae.

Thirty-four isolates of Metarhizium spp. from Russian collections were genotyped using 5' EF-1α gene sequence analysis. Four species were identified, of which M. robertsii and M. brunneum were the most frequent, whereas M. anisopliae and M. pemphigum were sporadic. Radial growth studies in the temperature range of 10-40°C revealed that growth at high temperatures (35-37.5°C) was inherent for M. robertsii isolates but not for M. brunneum isolates. In contrast, M. brunneum isolates were more active at cold temperatures (10°C) compared to M. robertsii. Virulence was evaluated against larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, under two regimes: humid (21°C, 80% relative humidity (RH)) and arid (31°C, 55% RH). M. brunneum isolates were less virulent compared to M.robertsii under both regimes. M. robertsii activity did not differ under the two regimes, but M. brunneum was less virulent under the arid regime compared to the humid one. A field experiment under natural conditions (steppe zone of Western Siberia) with daily ranges of 10-43°C and 13-98% RH showed that M. robertsii was significantly more active than M. brunneum against CPB larvae.

Identification and Characterization of Pathogenic and Endophytic Fungal Species Associated with Pokkah Boeng Disease of Sugarcane.

Pokkah Boeng is a serious disease of sugarcane, which can lead to devastating yield losses in crop-producing regions, including southern China. However, there is still uncertainty about the causal agent of the disease. Our aim was to isolate and characterize the pathogen through morphological, physiological, and molecular analyses. We isolated sugarcane-colonizing fungi in Fujian, China. Isolated fungi were first assessed for their cell wall degrading enzyme capabilities, and five isolates were identified for further analysis. Internal transcribed spacer sequencing revealed that these five strains are Fusarium, Alternaria, Phoma, Phomopsis, and Epicoccum. The Fusarium isolate was further identified as F. verticillioides after Calmodulin and EF-1α gene sequencing and microscopic morphology study. Pathogenicity assay confirmed that F. verticillioides was directly responsible for disease on sugarcane. Co-inoculation of F. verticillioides with other isolated fungi did not lead to a significant difference in disease severity, refuting the idea that other cellulolytic fungi can increase disease severity as an endophyte. This is the first report characterizing pathogenic F. verticillioides on sugarcane in southern China.

Species of Metarhizium anisopliae complex implicated in human infections: retrospective sequencing study

ObjectivesFungi belonging to the Metarhizium anisopliae complex comprise ubiquitous arthropod pathogenic moulds used as mycopesticides. Rare cases of human infections due to M. anisopliae have been reported. We hypothesize misidentifications of fungal strains implicated in these cases or used in mycopesticides. MethodsA review of the literature was conducted to identify previously published cases. We collected some of these previous described strains and reported new cases, and a French mycopesticide containing M. anisopliae. All identifications were performed based on elongation factor-1α gene sequencing. ResultsWe report eight new cases of Metarhizium infection in humans (three from France and five from Australia). The strains isolated from these cases, and three others from already published cases and reported as M. anisopliae, were molecularly identified based on elongation factor-1α (Ef1-α) gene sequencing as follows: Metarhizium robertsii (six), Metarhizium guizhouense (three), Metarhizium brunneum (one) and Metarhizium pingshaense (one). ConclusionsIn this study, we report new human cases of Metarhizium infections, and, based on Ef-1α gene sequencing, we demonstrate the misidentification of species in case reports. We also correct the species identification of a strain reported as M. anisopliae used in a commercially available mycopesticide. According to our results, none of the strains from the human infection reports reviewed belongs to the species M. anisopliae.

Development of a PCR protocol for the identification and detection of Fusarium solani f. sp. piperis from soil and roots of black pepper (Piper nigrum)

The main disease of black pepper (Piper nigrum) in Brazil is caused by Fusarium solani f. sp. piperis. The disease diagnosis is problematic because, besides this pathogen, there are other different phylogenetic lineages within the Fusarium solani species complex (FSSC) associated with black pepper that can not be reliably identified based solely on morphological characteristics. Based on differences in the translation elongation factor 1-alpha (EF-1α) gene sequences of species of the FSSC, one pair of species-specific primers, Fpiper1F/Fpiper1R, was synthesized to accurately detect and identify F. solani f. sp. piperis. Primer specificity was tested with DNA from 18 isolates of F. solani f. sp. piperis, 24 representative isolates of other lineages of the FSSC associated with P. nigrum, and isolates representing eight strains of other species. The primers amplified only a single PCR band of 230 bp from F. solani f. sp. piperis. Nested PCR was more sensitive than conventional PCR, using the primer pair EF1/EF2 for the first round and specific primers Fpiper1F/Fpiper1R for the second round. Detection sensitivity was 1 fg of purified F. solani f. sp. piperis DNA template and 101 macroconidia/g soil infected. The specific primers will greatly facilitate the detection and identification of F. solani f. sp. piperis, and have potential as a diagnostic tool for detecting this pathogen in field-grown black pepper soil.

Isolation, characterization and virulence of entomopathogenic fungi from Gryllotalpa gryllotalpa (Orthoptera: Gryllotalpidae)

Mole crickets are significant pests of turf, vegetable and some tree seedlings worldwide, and it is costly to control them. In this study, we obtained 15 fungal isolates from Gryllotalpa gryllotalpa L. (Orthoptera: Gryllotalpidae) and compared the efficacy of these isolates against this pest with the aim of identifying their biocontrol potential. The fungal isolates were identified based on their morphological and molecular characteristics, using ITS, EF1-α, Bloc, RPB1, RPB2 and β-Tubulin gene sequencing. Consequently, the isolates were identified as Beauveria bassiana (Balsamo) Vuillemin (Gg-1), Clonostachys sp. (Gg-2, Gg-3, Gg-5, Gg-6, Gg-8 and Gg-13), Bionectria sp. (Gg-4), Metarhizium anisopliae (Metschnikoff) Sorokin (Gg-7, Gg-12 and Gg-14), Clonostachys rogersoniana (Schroers) (Gg-9 and Gg-15), Myriodontium sp. (Gg-10) and Myriodontium keratinophilum (Samson and Polon.) (Gg-11). These isolates caused mortalities ranging from 0 to 87 %, with the most virulent fungus being M. anisopliae Gg-12, which caused 87 % mortality within 15 days post inoculation using a conidial concentration of 1 × 107 conidia ml−1. Concentration–response test was conducted to determine the LC50 value of M. anisopliae Gg-12, and it was calculated as 1.069 × 106 conidia ml−1. As a result, M. anisopliae Gg-12 can be further investigated in terms of controlling mole crickets.