First Report of Lily Blight and Wilt Caused by Fusarium tricinctum in China

Lily (Lilium spp.) is one of the most well-known horticultural crops, and plays an important economic role in China. In September 2011, wilted plants were observed on Lilium oriental hybrid cultivar ‘Sorbonne’ growing in Longde County, Ningxia Hui Autonomous Region, China. Disease symptoms included wilting, stem and root rot, brown spots of bulbs and then bulbs rotting and spalling from the basal disc, plus a progressive yellowing and defoliation of the leaves from the base. Diseased plants were sampled from fields. Small pieces of symptomatic bulbs, stems, and roots from 10 different plants were surface disinfected with 75% ethanol for 30 s, 3% sodium hypochlorite for 5 min, and then washed three times in sterilize distilled water. The tissues were placed onto Martin Agar (2) at 25°C for 7 days. Nine isolates with morphology similar to Fusarium were obtained from the diseased tissues. Isolates were transferred to potato dextrose agar (PDA) and carnation leaf agar (CLA) and incubated at 25°C. Seven were identified as Fusarium oxysporum and one was F. solani, which have been reported as pathogens of lily in China (1). The other isolate, when grown on PDA, rapidly produced dense, white aerial mycelium that became pink with age and formed red pigments in the medium. On CLA, macroconidia with three to five septate were abundant, relatively slender, and curved to lunate. Microconidia were abundant, oval or pyriform, and one to two celled. Chlamydospores were in chains with smooth exine. The rDNA internal transcribed spacer (ITS) region and a portion of the translation elongation factor 1-alpha (EF-1α) gene of the fungus were amplified, with universal primers ITS1/ITS4 and EF1/EF2 primers respectively (3) and sequenced. In addition, the β-tubulin gene (β-tub) of the fungus was amplified with modified primers Btu-F-F01 (5′-CAGACMGGTCAGTGCGTAA-3′) and Btu-F-R01 (5′-TCTTGGGGTCGAACATCTG-3′) (4). BLASTn analysis showed that the ITS sequences of the isolate (GenBank Accession No. JX989827) had 98.9% similarity with those of F. tricinctum (EF611092, JF776665, and HM776425) and the EF-1α sequences of the isolate (JX989828) had 98.1% similarity with those of F. tricinctum (EU744837 and JX397850). The β-tub sequences of the isolate (JX989829) had 99.0% similarity with those of F. tricinctum (EU490236 and AB587077). The isolate was tested for pathogenicity. Two-month-old ‘Sorbonne’ seedlings were inoculated by placing 5 ml of conidial suspension (about 106 conidia per ml) over the roots of plants in each pot. Control plants were treated with sterile water in the same way. Plants were placed in a greenhouse at 22 to 25°C with a 15-h photoperiod. There were eight plants per pot and three replicates for each treatment. After 3 weeks, 87.5% of the inoculated plants exhibited browning of the root tips, root rot, and yellowing of the leaves, while control plants were symptomless. The pathogen was reisolated from the infected roots and identified as F. tricinctum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Fusarium wilt of lily caused by F. tricinctum. This information will provide guidance for the control of lily wilt disease and add information useful for the production of lilies. References: (1) C. Li and J. J. Li. Acta Phytopathol. Sin. (in Chinese) 26:192, 1995. (2) J. P. Martin. Soil Sci. 38:215, 1950. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. U. S. A. 95:2044, 1998. (4) M. Watanabe et al. BMC Evol. Biol. 11:322. 2011.

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First report of Fusarium falciforme (FSSC 3+4) causing root rot on chickpea in Mexico

Plant Disease ◽

10.1094/pdis-05-21-1123-pdn ◽

2021 ◽

Author(s):

Sixto Velarde Felix ◽

Victor Valenzuela ◽

Pedro Ortega ◽

Gustavo Fierros ◽

Pedro Rojas ◽

...

Keyword(s):

Fusarium Solani ◽

Root Rot ◽

Species Complex ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Chickpea (Cicer aretinium L.) is a legume crop of great importance worldwide. In January 2019, wilting symptoms on chickpea (stunted grow, withered leaves, root rot and wilted plants) were observed in three fields of Culiacan Sinaloa Mexico, with an incidence of 3 to 5%. To identify the cause, eighty symptomatic chickpea plants were sampled. Tissue from roots was plated on potato dextrose agar (PDA) medium. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Ff01 to Ff10). On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septae and light purple pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidias were falciform, hyaline, with slightly curved apexes, three to five septate, with well-developed foot cells and blunt apical cells, and measured 26.6 to 45.8 × 2.2 to 7.0 μm (n = 40). The microconidia (n = 40) were hyaline, one to two celled, produced in false heads that measured 7.4 to 20.1 (average 13.7) μm × 2.4 to 8.9 (average 5.3) μm (n = 40) at the tips of long monophialides, and were oval or reniform, with apexes rounded, 8.3 to 12.1 × 1.6 to 4.7 μm; chlamydospores were not evident. These characteristics fit those of the Fusarium solani (Mart.) Sacc. species complex, FSSC (Summerell et al. 2003). The internal transcribed spacer and the translation elongation factor 1 alpha (EF1-α) genes (O’Donnell et al. 1998) were amplified by polymerase chain reaction and sequenced from the isolate Ff02 and Ff08 (GenBank accession nos. KJ501093 and MN082369). Maximum likelihood analysis was carried out using the EF1-α sequences (KJ501093 and MN082369) from the Ff02 and Ff08 isolates and other species from the Fusarium solani species complex (FSSC). Phylogenetic analysis revealed the isolate most closely related with F. falciforme (100% bootstrap). For pathogenicity testing, a conidial suspension (1x106 conidia/ml) was prepared by harvesting spores from 10-days-old cultures on PDA. Twenty 2-week-old chickpea seedlings from two cultivars (P-2245 and WR-315) were inoculated by dipping roots into the conidial suspension for 20 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80% and a 12-h/12-h light/dark cycle. After 8 days, the first root rot symptoms were observed on inoculating seedlings and the infected plants eventually died within 3 to 4 weeks after inoculation. No symptoms were observed plants inoculated with sterilized distilled water. The fungus was reisolated from symptomatic tissues of inoculated plants and was identified by sequencing the partial EF1-α gene again and was identified as F. falciforme (FSSC 3 + 4) (O’Donnell et al. 2008) based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch’s postulates. The molecular identification was confirmed via BLAST on the FusariumID and Fusarium MLST databases. Although FSSC has been previously reported causing root rot in chickpea in USA, Chile, Spain, Cuba, Iran, Poland, Israel, Pakistan and Brazil, to our knowledge this is the first report of root rot in chickpea caused by F. falciforme in Mexico. This is important for chickpea producers and chickpea breeding programs.

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First Report of Nectria haematococca Causing Stem Rot of Soybean in China

Plant Disease ◽

10.1094/pdis-07-11-0626 ◽

2012 ◽

Vol 96 (3) ◽

pp. 457-457 ◽

Cited By ~ 1

Author(s):

Y. Gai ◽

R. Pan ◽

D. Xu ◽

M. Deng ◽

W. Chen ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Stem Rot ◽

Universal Primers ◽

Conidial Suspension ◽

Fruiting Bodies ◽

Nectria Haematococca ◽

First Report ◽

Soybean Seedlings

In October 2010, soybean (Glycine max) plants growing in commercial soybean fields in Zengcheng City, Guangdong Province developed symptoms consisting of stem and root rot, yellowing, and defoliation of leaves. Reddish, spherical fruiting bodies appeared in lesions that developed on stems. Plants with symptoms were sampled from fields. Fruiting bodies were excised from diseased tissues. Microscopic examination revealed that they were perithecia, globose to pyriform, and measured 197 to 260 μm in diameter and 226 to 358 μm long. When squeezed gently, cylindrical to clavate asci, 7.2 to 9.6 μm in diameter and 75.4 to 92.0 μm long, containing eight ascospores were exuded from the perithecia. Ascospores were ellipsoid to obovate, two celled, slightly constricted at the septum, had longitudinal striations, and measured 4.9 to 6.0 μm in diameter and 10.6 to 15.0 μm long. The fungus was isolated from the basal stem tissues of diseased soybean plants and cultured on potato dextrose agar (PDA) medium amended with streptomycin sulfate. On PDA, the culture developed into blue-pigmented colonies with whitish mycelium that produced oval to cylindrical microconidia. Microconidia had 0 to 1 septum, ranged from 2.5 to 5.2 × 7.6 to 29.4 μm, and were produced on monophialides. Macroconidia were cylindrical to falcate, thick walled, 2 to 5 septa, and 3.5 to 6.0 × 25.4 to 66.8 μm. Chlamydospores were present and ranged from 6.8 to 13.6 × 5.5 to 9.5 μm. Orange-to-reddish perithecia were readily formed in old culture. These morphological characteristics were consistent with descriptions of Nectria haematococca (anamorph Fusarium solani) (1). The rDNA internal transcribed spacer (ITS) region and the fragment of translation elongation factor 1-alpha (EF1-α) genes of the fungus were amplified, respectively, with universal primers ITS1/ITS4 and ef1/ef2 primers and sequenced. BLAST searches showed that the ITS sequences of three isolates (GenBank Accession Nos. JN015069, JN190942, and JN190943) had 99% similarity with those of N. haematococca(GenBank Accession Nos. DQ535186, DQ535185, and DQ535183) and the EF1-α sequences of three isolates (GenBank Accession Nos. JN874641, JN874642, and JN874643) had 100% similarity with those of F. solani (GenBank Accession Nos. DQ247265 and DQ247327). Completion of Koch's postulates confirmed the pathogenicity of the isolates in a replicated experiment. Thirty-day-old soybean seedlings of cultivar Huaxia No. 3 were inoculated by soaking their root systems in a conidial suspension (106 conidia per ml) for 30 min and then transplanted in plastic pots (20 cm in diameter) and incubated at 25 ± 2°C in a greenhouse. Control plants were treated with sterile water in the same way. There were four plants per pot and there were six replicates for each treatment. Within 3 weeks, more than 70% of the inoculated plants exhibited symptoms of leaf yellowing, stem rot, and root rots while control plants were symptomless. N. haematococca was reisolated from the diseased plants. To our knowledge, this is the first report of N. haematococca causing stem rot of soybean in China and the first description of sexual reproduction of F. solani causing soybean stem rot in nature. This pathogen may pose a serious threat to soybean production in China where soybean is a main crop. Reference: (1) C. Booth. The Genus Fusarium. CAB International, Wallingford, UK, 1971.

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First report of Diaporthe eres causing root rot of Coptis chinensis Franchet

Plant Disease ◽

10.1094/pdis-12-20-2564-pdn ◽

2021 ◽

Author(s):

PengYing MEI ◽

Xuhong Song ◽

Zhiyu Zhu ◽

Longyun Li

Keyword(s):

Root Rot ◽

Elongation Factor ◽

Its Region ◽

Small Samples ◽

Molecular Characteristics ◽

Conidial Suspension ◽

Coptis Chinensis ◽

Base Pairs ◽

First Report ◽

Surface Sterilization

Chongqing coptis (Coptis chinensis Franchet) industry produces more than 60% of the Chinese coptis crop, and has been exported to many countries and regions. Since 2008, root rot has become a serious and widespread disease on coptis plants in Shizhu county with an average incidence of 40%, and yield losses up to 67%. Symptomatic coptis plants showed stunted growth, with the fibrous roots and main roots having brown or black, rotten, necrotic lesions. To our knowledge, Fusarium solani, F. carminascens, F. oxysporum and F. tricinctum have been previously reported as pathogens of coptis root rot (Luo et al. 2014; Cheng et al. 2020; Wu et al. 2020), but non Fusarium pathogens has not been reported yet. In order to identify new pathogens, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E) in October 2019. Small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, and then put on PDA after surface sterilization. Cultures were incubated at 25°C in dark until fungal colonies were observed. After subculturing for 3 times, 3 out of 21 isolates yielded a similar type of fungal colony. White, aerial, fluffy mycelium were formed and reached 8.3 cm diameter within 7 days, and dark pigmentation developed in the centre. Colonies turned to gray with age, and abundant dark brown pycnidia and black stromata were formed at maturity. Alpha conidia were aseptate, hyaline, fusiform to ellipsoidal, often biguttulate, measuring (6.0-8.5)×(2.0-3.0) μm. Beta conidia were aseptate, hyaline, linear to hooked, measuring (18-30)×(1.0-1.5) μm (Figure S1). For further identification, a multigene phylogenetic analysis was carried out. The internal transcribed spacer (ITS), translation elongation factor 1ɑ (tef1-ɑ), histone H3 (his3), calmodulin (cal), and β-tubulin (tub2) gene regions were amplified with ITS1/ITS4, EF1-728F/EF1-986R, CYLH3F/H3-1b, CAL228F/CAL737R, T1/Bt2b (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Crous et al. 2004). GenBank accession numbers of isolate H13 were MT463391 for the ITS region, MT975573 for tef1-ɑ, MT975574 for his3, MT975575 for cal, and MT975576 for tub2. BLAST results showed the ITS, tef1-ɑ, his3, cal and tub2 sequences revealed 99.82% (553/554 base pairs), 100% (347/347 base pairs), 100% (474/474 base pairs), 99.39% (486/489 base pairs), and 99.14% (803/810 base pairs) homology respectively with those of Diaporthe eres (MN816416.1, KU557616.1, KC343564.1, KU557595.1, and KY569366.1). Thus, H13 were identified as D. eres based on its morphological and molecular characteristics. Pathogenicity of D. eres in coptis was investigated using the H13 isolate (1 of the 3 isolates). The roots of 10 healthy 2-year-old coptis plants were individually inoculated with 5 ml of a 106 conidia/mL conidial suspension and sterilized water was used to mock inoculate. Thirty days after inoculation, most of the inoculated coptis roots showed dark brown and rotten root, similar to those observed in the field, whereas mock inoculated roots showed healthy. D. eres was recovered from symptomatic roots and identified based on morphology. To our knowledge, this is the first report of D. eres causing root rot of coptis not only in China but anywhere in the world.

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First report of seedling blight of maize caused by Fusarium asiaticum in Northeast China

Plant Disease ◽

10.1094/pdis-08-20-1727-pdn ◽

2020 ◽

Author(s):

Huaiyu Dong ◽

Peiwen Qin ◽

Zenggui Gao ◽

Jing Xu ◽

Xiude Xu

Keyword(s):

Northeast China ◽

Seedling Emergence ◽

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Liaoning Province ◽

Conidial Suspension ◽

Seedling Blight ◽

First Report ◽

Fusarium Asiaticum

Maize [Zea mays L.] is an important food and feed crops in northeast of China. In 2019, maize seedling blight with an incidence of up to 25% was found at the field in Fushun city of Liaoning Province. Typical symptoms of seedlings were yellow, thin, wilt and die. The leaves gradually became yellow from the base of the plant to the top. Root system was poorly developed. The primary roots were usually discolored and rotted. And faintly pink or puce-coloured mould was found on seeds of the rotted seedings. Symptomatic roots of diseased seedling were collected and surface-disinfested with 70% ethanol for 1 min and then in 2% NaClO for 3 min, rinsed with sterilized water three times, cut into small pieces and placed on potato dextrose agar (PDA) medium for 5 days at 25 °C. Colonies on PDA were pink to dark red with fluffy aerial mycelium and red to aubergine pigmentation with the age. The causal agent was transferred to carnation leaf agar (CLA) medium and incubated at 25°C under a 12-h light-dark cycle. 12 Pure cultures were obtained from single conidia with an inoculation needle under stereomicroscope. The harvested macroconidia were hyaline, falcate with single foot cells, 3–5 septate and 28.2- 43.5 μm × 3.7 - 4.9 μm. Chlamydospores were globose to subglobose (5 to 13.5 μm). No microconidia were found. The perithecia were black, ostiolate subglobose. Asci were hyaline, clavate, measuring 58.1- 83.9 µm × 7.7- 11.9 µm and contained eight ascospores. Morphological characters of the pathogen agreed well with descriptions of Fusarium asiaticum (O’Donnell et al.2004; Leslie and Summerell 2006). To confirm the identity, partial translation elongation factor 1 alpha (TEF1-a) gene and rDNA internal transcribed spacer (ITS) region of isolate MSBL-4 were amplified and sequenced (O’Donnell et al. 2015; White et al.1990). BLASTn analysis of both TEF sequence (MT330257) and ITS sequence (MT322117), revealed 100% sequence identity with F. asiaticum KT380116 and KX527878, respectively. The isolate MSBL-4 was NIV chemotype as determined by Tri13F/DON, Tri13NIV/R (Chandler et al, 2003) assays. Pathogenicity studies were conducted on maize hybrid "Liaodan 565". Inoculum of F. asiaticum was prepared from the culture of MSBL-4 incubate in 2% mung beans juice on a shaker (150 rpm) at 25°C for 48 hours. The five liter pots (10 pots) were filled with sterilized field soil and five of them were mixed with conidial suspension (300mL in each pot) at 2 × 105 conidia per ml. Ten kernels per pot were surface disinfected in 2% sodium hypochlorite for 5 min, rinsed with sterilized water and planted. Five pots were inoculated and another uninoculated five pots served as controls. The pots were maintained in a greenhouse at 22-26°C for 40 days. Leaves of the plants in inoculated pots were yellowing and the roots became discolored or necrotic rot at 4 weeks after seedling emergence. All characteristics of the disease were similar to those observed in field. Non-inoculated control plants had no symptoms. Fusarium asiaticum was reisolated from inoculated plants and was identical to the original isolate. The experiment was repeated once with similar results. To our knowledge, this is the first report of seedling blight caused by F. asiaticum on maize in northeast China, and it has posed a threat to maize production of China. References: Leslie J F and Summerell BA. 2006. The Fusarium laboratory manual. Blackwell Publishing, Ames, pp 176-179. O’Donnell et al.2004. Fungal Genetics and Biology 41: 600-623. O’ Donnell et al. 2015. Phytoparasitica 43:583-595. White T J et al. 1990. Academic Press, San Diego, CA, pp 315-322. Chandler E A et al. 2003. Physiological and Molecular Plant Pathology 62(6): 355–367.

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First Report of Fusarium stem and root rot of Gerbera jamesonii caused by Fusarium incarnatum in China

Plant Disease ◽

10.1094/pdis-02-21-0440-pdn ◽

2021 ◽

Author(s):

Shuning Chen ◽

Wei Sun ◽

Huizhu Yuan ◽

Xiaojing Yan

Keyword(s):

Root Rot ◽

Vascular System ◽

Elongation Factor ◽

Morphological Observation ◽

Conidial Suspension ◽

Gerbera Jamesonii ◽

Gene Sequences ◽

Plastic Container ◽

First Report ◽

Brown Discoloration

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 as Fusarium incarnatum based 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.

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First Report of Euphorbia larica Dieback Caused by Fusarium brachygibbosum in Oman

Plant Disease ◽

10.1094/pdis-09-12-0828-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 687-687 ◽

Cited By ~ 9

Author(s):

I. H. Al-Mahmooli ◽

Y. S. Al-Bahri ◽

A. M. Al-Sadi ◽

M. L. Deadman

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Common Component ◽

First Report ◽

Ethnobotanical Uses ◽

Artificial Inoculations ◽

Stem Lesions ◽

Desert Flora

Euphorbia larica Boiss. (Arabic = Isbaq) is a dominant and common component of the native desert flora of northern Oman. Traditional ethnobotanical uses have included use of the latex for treating camels with parasites. In February 2011, E. larica plants showing stem lesions up to several cm long and in many cases with stem dieback were collected from Al-Khoudh 50 km west of Muscat. The disease appeared widespread within the location where several dead specimens were also recorded, although the cause was unclear. Sections (5 mm) of five diseased branches taken from different plants and placed on potato dextrose agar (PDA) in all cases yielded Fusarium-like colonies. Colonies recovered were initially white becoming rose to medium red in color with abundant aerial mycelium. Macroconidia were scarce and scattered (mean of 20 spores: 26.83 × 4.73 μm) with three to four septa per spore; microconidia were slightly curved, ovoid, and fusiform (mean of 20 spores: 11.64 × 4.03 μm) with zero to two septa per spore. Spherical chlamydospores (mean of 20 spores: 11.05 μm) were terminal and intercalary, single, and in chains. In vitro characters and spores measurements conformed to previously described features of Fusarium brachygibbosum Padwick (1). Mycelial plugs (5 mm) were taken from 7-day-old cultures of the fungus grown on 2.5% PDA and applied to a small incision (3 mm) on the stems of healthy E. larica grown in situ and protected with wet cotton and Parafilm. The residual agar, mycelium, cotton, and Parafilm were removed after 7 days and symptoms were recorded. Control stems were inoculated using PDA (5 mm) plugs alone and inoculations were repeated twice. Artificial inoculations resulted in dieback of all stems within 11 days and fungal colonies identical to initial isolations were recovered from artificially infected surface-sterilized stem pieces. Identification of F. brachygibbosum was confirmed by comparing sequences generated from the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1 and ITS4 primers) and the intron region of translation elongation factor alpha (EF1-α) (EF-1-986 and EF-728 primers). The ITS and EF1-α sequences were found to share 100% and 99% nucleotide similarity to previously published sequences of the ITS (HQ443206) and EF1-α (JQ429370) regions of F. brachygibbosum in GenBank. The accession number of ITS sequence of one isolate assigned to EMBL-Bank was HF562936. The EF sequence was assigned to EMBL-Bank accession (submission number Hx2000027017; number will be sent later). This pathogen has previously been reported on date palm (2) in Oman but, to our knowledge, this is the first report of this pathogen on E. larica. References: (1) A. M. Al-Sadi et al. Crop Prot. 37:1, 2012. (2) G. W. Padwick. Mycol. Pap. 12:11, 1945.

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First report of Trichoderma afroharzianum causing seed rot on maize in Italy

Plant Disease ◽

10.1094/pdis-12-21-2697-pdn ◽

2022 ◽

Author(s):

Martina Sanna ◽

Massimo Pugliese ◽

Maria Lodovica GULLINO ◽

Monica Mezzalama

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Aerial Mycelium ◽

Light Cycle ◽

Conidial Suspension ◽

Ear Rot ◽

Trichoderma Spp ◽

Trichoderma Species ◽

First Report ◽

Pathogenicity Tests

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 60,602,320 t, covering 588,597 ha (ISTAT 2021). Trichoderma species are widespread filamentous fungi in soil, well known and studied as biological control agents (Vinale et al., 2008). Seeds of a yellow grain hybrid (class FAO 700, 132 days) were collected in September 2020 from an experimental field located in Carmagnola (TO, Italy: GPS: 44°53'11.0"N 7°40'60.0"E) and tested with blotter test (Warham et al., 1996) to assess their phytosanitary condition. Over the 400 seeds tested, more than 50% showed rotting and development of green mycelium typical of the genus Trichoderma. Due to the high and unexpected percentage of decaying kernels, ten colonies were identified by morphological and molecular methods. Single conidia colonies of one Trichoderma (T5.1) strain were cultured on Potato Dextrose Agar (PDA) for pathogenicity tests, and on PDA and Synthetic Nutrient-Poor Agar (SNA) for morphological and molecular identification. The colonies grown on PDA and SNA showed green, abundant, cottony, and radiating aerial mycelium, and yellow pigmentation on the reverse. Colony radius after 72 h at 30°C was of 60-65 mm on PDA and of 50-55 mm on SNA. The isolates produced one cell conidia 2.8 - 3.8 µm long and 2.1 - 3.6 µm wide (n=50) on SNA. Conidiophores and phialides were lageniform to ampulliform and measured 4.5 – 9.7 µm long and 1.6 – 3.6 µm wide (n=50); the base measure 1.5 – 2.9 µm wide and the supporting cell 1.4 – 2.8 µm wide (n=50). The identity of one single-conidia strain was confirmed by sequence comparison of the internal transcribed spacer (ITS), the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (Oskiera et al., 2015). BLASTn searches of GenBank using ITS (OL691534) the partial tef-1α (OL743117) and rpb2 (OL743116) sequences of the representative isolate T5.1, revealed 100% identity for rpb2 to T. afroharzianum TRS835 (KP009149) and 100% identity for tef-1α to T. afroharzianum Z19 (KR911897). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile H2O to 1×106 CFU/ml. Twenty-five seeds were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 23°C under a seasonal day/night light cycle. Twenty primary ears were inoculated, by injection into the silk channel, with 1 ml of a conidial suspension of strain T5.1 seven days after silk channel emergence (BBCH 65) (Pfordt et al., 2020). Ears were removed four weeks after inoculation and disease severity, reaching up to 75% of the kernels of the twenty cobs, was assessed visually according to the EPPO guidelines (EPPO, 2015). Five control cobs, inoculated with 1 ml of sterile distilled water were healthy. T. afroharzianum was reisolated from kernels showing a green mold developing on their surface and identified by resequencing of tef-1α gene. T. afroharzianum has been already reported on maize in Germany and France as causal agent of ear rot of maize (Pfordt et al. 2020). Although several species of Trichoderma are known to be beneficial microorganisms, our results support other findings that report Trichoderma spp. causing ear rot on maize in tropical and subtropical areas of the world (Munkvold and White, 2016). The potential production of mycotoxins and the losses that can be caused by the pathogen during post-harvest need to be explored. To our knowledge this is the first report of T. afroharzianum as a pathogen of maize in Italy.

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First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Plant Disease ◽

10.1094/pdis-11-20-2471-pdn ◽

2021 ◽

Author(s):

Xiujing Hong ◽

Shijia Chen ◽

linchao Wang ◽

Bo Liu ◽

Yuruo Yang ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Average Diameter ◽

Ethanol Solution ◽

Likelihood Method ◽

Conidial Suspension ◽

Multiple Sequence ◽

First Report ◽

Pure Cultures

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

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First Report of Stem Dieback Caused by Lasiodiplodia parva on Styphnolobium japonicum in China

Plant Disease ◽

10.1094/pdis-08-21-1598-pdn ◽

2021 ◽

Author(s):

Ziwei Zhou ◽

Cuiping Wu ◽

Jing Yang ◽

Jieying Xu ◽

Zhenpeng Chen ◽

...

Keyword(s):

Robinia Pseudoacacia ◽

Elongation Factor ◽

Aerial Mycelium ◽

Random Trees ◽

Molecular Evidence ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Accurate Identification ◽

Individual Tree ◽

First Report

Styphnolobium japonicum (L.) Schott is a variant of Robinia pseudoacacia and is a popular Asian tree widely used in traditional medicine. From March 2019 to 2021, a disease was found on the campus of Nanjing Forestry University and several landscape sites of Xuanwuhu Park, causing dieback. Most of the trees (approximately 40%) have rotted branches. On average, 60% of the branches per individual tree were affected by this disease. The initial round lesions were grayish brown. In the later stage, the whole branch becomes black and produces spherical fruiting bodies . Twenty diseased branches were picked from three random trees. Small tissues (3-4mm²) were surface-sterilized in 75% ethanol for 30 s followed by 1% NaClO for 90 s and placed on potato dextrose agar (PDA), and incubated in the dark at 25°C for three days. Hyphae were visibly emerged from 70% of the samples. Three representative isolates (Lth-soj1, Lth-soj2, and Lth-soj3) were obtained and deposited in China’s Forestry Culture Collection Center (Lth-soj1: cfcc55896, Lth-soj2: cfcc55897, Lth-soj3: cfcc55898). The colonies of three isolates on PDA were fast growing and white, which turned grey to dark grey after 3 days of incubation in the dark at 28°C . Two-weeks old colonies were black and fluffy on PDA, with abundant aerial mycelium, and the reverse side too was black in color. The fungus usually grew well on PDA and produced pycnidia and conidia within 3–4 weeks. Conidia were initially hyaline and aseptate, ellipsoid to ovoid, with granular content, apex broadly rounded, remaining hyaline and later becoming dark brown, one septate, thick walled, base truncate or round and longitudinally striate. The conidia (n=30) of a representative isolate（Lth-soj1）, measured 24.3 ± 0.3 μm in length and 13.3 ± 0.5 μm in width . The morphological characters of the three isolates matched those of Lasiodiplodia parva(Alves et al. 2008). For accurate identification, the DNA of the three isolates was extracted. The internal transcribed spacer region (ITS), translation elongation factor (EF1-α), and β-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/ITS4 , EF1-728F/EF1-986R, and Bt2a/Bt2b , respectively. The sequences were deposited in GenBank under accession numbers MZ613154, MZ643245 and MZ643242 for Lth-soj1, MZ613155, MZ643246 and MZ643244 for Lth-soj2, and MZ613157, MZ643247 and MZ643243 for Lth-soj3. The ITS, EF1-α, and TUB2 sequences of isolate Lth-soj1 (GenBank Acc. No. MZ613154, MZ643245, MZ643242) were 100% (519/519 nt), 99.34% (299/301 nt), and 99.77% (436/437 nt) identical to those of MZ182360, EF622063, and MK294119, respectively. Interspecific differences were observed in a maximum-likelihood tree of Lasiodiplodia species using the concatenated dataset. Based on the morphological and molecular evidence, the isolates were identified as L. parva. The pathogenicity of three isolates were tested on potted three-year-old seedlings (100-cm tall) of S. japonicum maintained in a greenhouse. Healthy stems were wounded with a sterile needle then inoculated with 10 µL of conidial suspension. Control plants were treated with ddH2O. In total, 12 seedlings were inoculated including three controls. Three seedlings per isolate and 10 stems per seedling were used for each treatment. The plants were kept inside sealed polythene bags for the first 24 h and sterilized H2O was sprayed into the bags twice a day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25/16°C. Within seven days, all the inoculated points showed lesions similar to those observed in field and the conidiomatas growing on the surface of the branches, whereas controls were asymptomatic . The infection rate of each of the three isolates was 100%. The strain was re-isolated from the lesions and sequenced as L.parva, whereas not from control stems. This is the first report of L. parva causing rotten branches of S. japonicum in China and the worldwide. These data will help to develop effective strategies for managing this newly emerging disease.

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First Report of Lasiodiplodia theobromae Associated with Decline of Grapevine Rootstock Mother Plants in Spain

Plant Disease ◽

10.1094/pdis-92-5-0832b ◽

2008 ◽

Vol 92 (5) ◽

pp. 832-832 ◽

Cited By ~ 13

Author(s):

A. Aroca ◽

R. Raposo ◽

D. Gramaje ◽

J. Armengol ◽

S. Martos ◽

...

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Pathogenic Fungi ◽

Vascular Lesions ◽

Malt Extract ◽

Pathogenicity Test ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Mother Plants

A field of Richter 110 rootstock mother plants in Valencia Province (eastern Spain) was surveyed during November 2006 to study the mycoflora of declining plants. Two canes with stunted leaves were collected from a plant with a reduced number of shoots. No cankers or vascular lesions were observed in the collected canes. Six wood chips (1 to 2 mm thick) were taken from one basal fragment (3 to 4 cm long) of each cane, surface sterilized in 70% ethanol for 1 min, and plated on malt extract agar supplemented with 0.5 g L–1 of streptomycin sulfate. Petri dishes were incubated for 7 days at 25°C. A fungus was consistently isolated from all samples that showed the following characteristics: colonies grown on potato dextrose agar (PDA) at 25°C developed a white, aerial mycelium that turned gray after 4 to 6 days and produced pycnidia after 1 month on sterile grapevine slivers of twigs placed on the PDA surface; conidia from culture were ellipsoidal, thick walled, initially hyaline, nonseptate, and measuring 20 to 25 (22.5) × 12 to 14 (13) μm; aged conidia were brown, 1-septate with longitudinal striations in the wall; and pseudoparaphyses variable in form and length were interspersed within the fertile tissue. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the above characteristics (2). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) region from the rRNA repeat and part of the translation elongation factor 1-alpha (EF1-α) and the β-tubulin (B-tub) genes, as done elsewhere (1,3). BLAST searches at GenBank showed a high identity with reference sequences (ITS: 100%, EF1-α: 97%; B-tub: 99%). Representative sequences of the studied DNA regions were deposited at GenBank (Accession Nos.: ITS: EU254718; EF1-α: EU254719; and B-tub: EU254720). A pathogenicity test was conducted on 1-year-old grapevine plants cv. Macabeo grafted onto Richter 110 rootstocks maintained in a greenhouse. A superficial wound was made on the bark of 10 plants with a sterilized scalpel, ≈10 cm above the graft union. A mycelial plug obtained from the margin of an actively growing fungal colony (isolate JL664) was placed in the wound and the wound was wrapped with Parafilm. Ten additional control plants were inoculated with sterile PDA plugs. All control plants grew normally, and the inoculation wound healed 3 months after inoculation. Plants inoculated with L. theobromae showed no foliar symptoms in the same period, but developed cankers variable in size surrounding the inoculation sites. Vascular necroses measuring 8.4 ± 1.5 cm (mean ± standard error) developed in the inoculated plants that were significantly longer than the controls (0.3 ± 0.2 cm). The pathogen was reisolated from all inoculated plants and no fungus was reisolated from the controls. These results confirmed the pathogenicity of L. theobromae to grapevine and points to a possible involvement of L. theobromae in the aetiology of grapevine decline as previously reported (3,4). To our knowledge, this is the first report of L. theobromae isolated from grapevine in Spain. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45(suppl.):S43, 2006.

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