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

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 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 Fusarium equiseti Causing Fusarium Wilt on Potato (Solanum tuberosum) in China

Plant Disease ◽

10.1094/pdis-02-21-0281-pdn ◽

2021 ◽

Author(s):

Lingxiao Cui ◽

Chengde Yang ◽

Liping Yang ◽

Mengjun Jin ◽

Lijuan Wei

Keyword(s):

Solanum Tuberosum ◽

Rna Polymerase Ii ◽

Fusarium Wilt ◽

Elongation Factor ◽

Morphological Characteristics ◽

Gansu Province ◽

Vascular Bundles ◽

First Report ◽

Potato Plants ◽

Pathogenicity Tests

Potato (Solanum tuberosum) is one of the most economically important crops in China, containing carbohydrates, protein, fiber, numerous vitamins and minerals, and is a heart healthy food (Raidl, 2020). Potato infected by Fusarium spp. exhibits quality and yield decline, and even death. In infected plants, the upper leaves exhibit chlorosis, the lower leaves wither and the vascular bundles of stems and tubers turn yellow, and then tan to brown. In August 2018, symptomatic potato stems and roots were collected from Zhangye city, Gansu province, China. Diseased stem tissues were surface sterilized with 75% alcohol for 30 s, and then rinsed in sterile water. The tissue pieces were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. Fusarium-like colonies were consistently isolated and three monoconidial isolates were obtained. Isolate 3SMJ-2 was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. 3SMJ-2 was inoculated in PDA liquid medium, grown on a shaker for 7 days at 25℃ to obtain a mix suspension of hypha fragments and spores (107 spores/mL). Healthy potato plants, named “Xin Daping” and were planted in pots (17 cm diameter by 12 cm) filled with 2L of sterile soil per pot. After 8 weeks, the plants were inoculated with the inoculum or distilled water. Then they were incubated in growth chambers at 25°C under a 12-h/12-h day/night potato period with 90% relative humidity for 24 h. For each treatment, 3 pots were inoculated. After 50 days, 100% of the inoculated potato plants exhibited wilt symptoms similar to those in the field but the control plants were symptomless. A Fusarium identical to strain 3SMJ-2 was re-isolated from symptomatic potato plants to fulfilling Koch’s postulates. Morphological characteristics of the re-isolated strain were identical to the original isolate, which confirmed pathogenicity of strain 3SMJ-2 originally isolated from the potatoes. Colonies of 3SMJ-2 were white with short conidiophores, a few microconidia and sickle-shaped macroconidia (25.2 to 42.9× 3.1 to 4.6 µm) (n = 60) with 4~7 septa, and mostly 5 septa, after cultivated on PDA in an incubator at 25℃ for 14 days. Spherical terminal or intercalary chlamydospores were observed on the mycelium. Strain 3SMJ-2 was identified preliminarily as Fusarium sp. based on morphological characteristics (Leslie et al., 2006). Genomic DNA was extracted from 3SMJ-2 using the OMEGA Fungal DNA kit according to the manufacturer’s protocol. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF) and RNA polymerase II second largest subunit (RPB2) were amplified using ITS1/ITS4 (White et al., 1990), Ef728M/Tef1R (Stępień et al., 2012) and 5F2 /7cR (O'Donnell et al., 2007), respectively. After sequencing by Beijing TSINGKE Biological Technology Co., Ltd., 3 fragments of approximately 519 bp, 587 bp and 1059 bp from the strain 3SMJ-2 were deposited in GenBank as MN420681, MW561963 and MW561964. The ITS, TEF and RPB2 sequences were 100%, 100% and 99.8% identical to those of F.equiseti (KY365589, KF499577, and MH582110). Based on the pathogenicity tests, morphological characteristics and molecular analyses, we identified the strain 3SMJ-2 as F. equiseti, the pathogen causing Fusarium wilt on potato in Zhangye City. Although, F. equiseti has been reported to cause root rot of cowpea (Li et al., 2017) and sugar beet (Cao et al., 2018) in China. To our knowledge, this is the first report confirming F. equiseti causing potato wilt in China. Potato is an economically important crop in Gansu and the occurrence of the new disease caused by F. equiseti on potato needs to be properly managed to reduce yield loss.

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First Report of Fusarium Maize Ear Rot Caused by Fusarium meridionale in China

Plant Disease ◽

10.1094/pdis-01-14-0108-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1156-1156 ◽

Cited By ~ 4

Author(s):

H. Zhang ◽

W. Luo ◽

Y. Pan ◽

J. Xu ◽

J. S. Xu ◽

...

Keyword(s):

Fusarium Species ◽

Elongation Factor ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Fusarium Ear Rot ◽

Ear Rot ◽

Translation Elongation Factor ◽

Translation Elongation ◽

First Report

Fusarium is an important genus of fungal pathogens that are responsible for devastating diseases, such as Fusarium ear rot on maize, which may result in yield losses and/or mycotoxin contamination. In September 2013, a survey to determine population composition of Fusarium species on maize was conducted at 22 fields in 18 counties in Gansu Province. Maize ears with clear symptoms (with a white to pink- or salmon-colored mold at the ear tip) were collected. Symptomatic seeds were surface-sterilized with 70% ethanol and 10% sodium hypochlorite and rinsed three times with sterile water to eliminate hypochlorite residues. After drying on sterile filter paper, the seeds were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 3 days. Mycelium that was characteristic of Fusarium spp. (2) was purified by transferring single spores to fresh PDA. Fusarium species were identified by morphological characteristics (2), multilocus genotyping assay (MLGT) (3), and sequence analysis of the translation elongation factor-1α (TEF) gene. Several Fusarium species were identified and Fusarium verticillioides and F. proliferatum were the predominant species. Based on MLGT, two strains from Chenghong County were identified as F. meridionale with NIV chemotype, a species in F. graminearum species complex (FGSC). Morphological characteristics were also identical to FGSC. Colonies grew rapidly on PDA and produce relatively large amounts of dense mycelia and red pigments. Slender, thick-walled, and moderately curved or straight macroconidia were observed with 5- to 6-septate. Furthermore, conidia on SNA also showed typical characteristics of F. meridionale, as the dorsal and ventral lines were often parallel and gradually curved. Sequences comparison of the partial translation elongation factor (TEF-1α, 644 bp) gene (1) was used to validate these observations. BLASTn analysis with the FUSARIUM-ID database revealed 100% sequence identity to F. meridionale (GenBank Accession No. KJ137017). Thus, both morphological and molecular criteria supported identification of the strains as F. meridionale. A pathogenicity test was performed on Zhengdan958, the maize variety with the largest planted acreage in China. Four days after silk emergence, 2 ml conidial suspension (105 macroconidia/ml) of each isolate were injected into each of 10 maize ears through silk channel. Control plants were inoculated with sterile distilled water. Typical FER symptoms (reddish-white mold) was observed on inoculated ears and no symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the same fungus from the infected seeds. F. meridionale was one of the pathogens causing Fusarium head blight on wheat and barley in China and produced nivalenol (4,5) and it also has been isolated from maize in Korea and Nepal. To our knowledge, this is the first report of F. meridionale causing Fusarium ear rot on maize in China. Further studies on biological characteristics such as temperature sensibility and fungicide resistance are needed to gain a better understanding of this new pathogen. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008. (4) L. Yang et al. Phytopathology 98:719, 2008. (5) H. Zhang et al. Plos one 7:e31722, 2012.

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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 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 Anthracnose Stalk Rot of Maize Caused by Colletotrichum graminicola in Switzerland

Plant Disease ◽

10.1094/pdis-09-13-0912-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 694-694

Author(s):

S. A. Sukno ◽

J. M. Sanz-Martín ◽

M. González-Fuente ◽

J. Hiltbrunner ◽

M. R. Thon

Keyword(s):

Average Length ◽

Aerial Mycelium ◽

Universal Primers ◽

Light Cycle ◽

Conidial Suspension ◽

Colletotrichum Graminicola ◽

Tissue Samples ◽

Average Width ◽

First Report ◽

Representative Sequence

Maize stem samples exhibiting symptoms of anthracnose were collected from a field near Zurich, Switzerland, in September of 2012 and were sent to the fungal genetics laboratory, Centro Hispano-Luso de Investigaciones Agrarias (CIALE) at the University of Salamanca, Spain, for further analysis. The stem samples exhibited glossy, black, and irregularly shaped lesions. Tissue samples, approximately 5 mm2, were dissected from below the epidermis. The tissue samples were surface disinfested for 1 min in 20% sodium hypochlorite and cultured on one half strength acidified PDA supplemented with ampicillin (2). Monoconidial isolates from three different stems were grown on potato dextrose agar (PDA) and had dark gray aerial mycelium with orange spore masses. Conidia were falcate, slightly curved, tapered toward the tips with an average length of 31.77 μm and an average width of 4.76 μm and produced in acervuli with setae, consistent with descriptions of C. graminicola Ces. Wils. Conidial suspensions were prepared for each isolate, and were inoculated onto the leaves of 2-week-old maize plants by laying the plants horizontally in a tray (in pots with their root systems intact) and placing 7.5-μl droplets of a 106 conidial suspension on the leaf surface. The trays were covered and plants were incubated overnight at 23°C. The plants were then returned to their upright position and grown in a growth chamber at 25°C with a 12-h light cycle (3). After 6 days, the inoculated plant leaves exhibited lesions that were elongated and irregularly shaped with necrotic centers and chlorotic margins. The water-inoculated controls did not show symptoms. Microscopic examination revealed the production of conidia on the surface of the leaves, identical to the original isolates. Genomic DNA was extracted using the protocol of Baek and Kenerley (1). A region of the ribosomal DNA repeat was amplified and sequenced using the universal primers ITS4 and ITS5. The resulting sequences were 100% identical to each other and 100% identical to C. graminicola sequences in GenBank. One representative sequence was deposited in GenBank under accession no. KF597538. The 100 most similar sequences in GenBank were used to construct a phylogenetic tree using the neighbor-joining method. The phylogenetic analysis revealed that the isolates clustered within the C. graminicola clade, consistent with their identification as C. graminicola. To our knowledge, this is the first report of anthracnose on maize caused by C. graminicola in Switzerland. Previous reports have demonstrated that the pathogen exists in neighboring countries Germany and France. References: (1) J.-M. Baek and C. M. Kenerley. Fungal Genet. Biol. 23:34, 1998. (2) S. A. Sukno et al. Appl. Environ. Microbiol. 74:823, 2008. (3) W. A. Vargas et al. Plant Physiol. 158:1342, 2012.

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First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky

Plant Disease ◽

10.1094/pdis-06-21-1292-pdn ◽

2021 ◽

Author(s):

Gabdiel Yulfo-Soto ◽

Henry S Smith ◽

Desiree Szarka ◽

Ed Dixon ◽

Lisa Vaillancourt ◽

...

Keyword(s):

Fusarium Graminearum ◽

Cannabis Sativa ◽

Elongation Factor ◽

Fungal Growth ◽

Tween 20 ◽

Conidial Suspension ◽

Ear Rot ◽

Head Blight ◽

Dark Cycle ◽

First Report

In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar ‘White CBG’ being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars ‘Lifter’, ‘Trump Towers’, ‘Wife’ and ‘White CBG’ were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O’Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.

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

Plant Disease ◽

10.1094/pdis-11-12-1010-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 993-993 ◽

Cited By ~ 10

Author(s):

Y. Y. Li ◽

Y. J. Wang ◽

Z. K. Xie ◽

R. Y. Wang ◽

Y. Qiu ◽

...

Keyword(s):

Root Rot ◽

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Universal Primers ◽

Conidial Suspension ◽

Root Tips ◽

First Report ◽

Horticultural Crops ◽

Blastn Analysis

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 Anthracnose of Papaya (Carica papaya L.) Caused by Colletotrichum siamense in China

Plant Disease ◽

10.1094/pdis-10-20-2154-pdn ◽

2021 ◽

Author(s):

Yujie Zhang ◽

Wenxiu Sun ◽

Ping Ning ◽

Tangxun Guo ◽

SuiPing Huang ◽

...

Keyword(s):

Carica Papaya ◽

Disease Incidence ◽

Aerial Mycelium ◽

Postharvest Disease ◽

Distilled Water ◽

First Report ◽

Surface Samples ◽

Initial Symptoms ◽

Colletotrichum Siamense ◽

Pathogenicity Tests

Papaya (Carica papaya L.) is a rosaceous plant widely grown in China, which is economically important. Anthracnose caused by Colletotrichum sp. is an important postharvest disease, which severely affects the quality of papaya fruits (Liu et al., 2019). During April 2020, some mature papaya fruits with typical anthracnose symptoms were observed in Fusui, Nanning, Guangxi, China with an average of 30% disease incidence (DI) and over 60% DI in some orchards. Initial symptoms of these papayas appeared as watery lesions, which turned dark brown, sunken, with a conidial mass appearing on the lesions under humid and warm conditions. The disease severity varied among fruits, with some showing tiny light brown spots, and some ripe fruits presenting brownish, rounded, necrotic and depressed lesions over part of their surface. Samples from two papaya plantations (107.54°E, 22.38°N) were collected, and brought to the laboratory. Symptomatic diseased tissues were cut into 5 × 5 mm pieces, surface sterilized with 2% (v/v) sodium hypochlorite for 1 minute, and rinsed three times with sterilized water. The pieces were then placed on potato dextrose agar (PDA). After incubation at 25°C in the dark for one week, colonies with uniform morphology were obtained. The aerial mycelium on PDA was white on top side, and concentric rings of salmon acervuli on the underside. A gelatinous layer of spores was observed on part of PDA plates after 7 days at 28°C. The conidia were elliptical, aseptate and hyaline (Zhang et al., 2020). The length and width of 60 conidia were measured for each of the two representative isolates, MG2-1 and MG3-1, and these averaged 13.10 × 5.11 μm and 14.45 × 5.95 μm. DNA was extracted from mycelia of these two isolates with the DNA secure Plant Kit (TIANGEN, Biotech, China). The internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS), β-tubulin 2 (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR and sequenced. The sequences were deposited into GenBank with accessions MT904003, MT904004, and MT898650 to MT898659. BLASTN analyses against the GenBank database showed that they all had over 99% identity to the type strain of Colletotrichum siamense isolate ICMP 18642 (GenBank accession numbers JX010278, GQ856775, JX009709, GQ856730, JX010410, JX010019) (Weir et al., 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS, TUB2 and GAPDH sequences using the Neighbor-joining algorithm also showed that the isolates were C. siamense. Pathogenicity tests were conducted on 24 mature, healthy and surface-sterilized papaya fruits. On 12 papaya fruits, three well separated wounded sites were made for inoculation, and for each wounded site, six adjacent pinhole wounds were made in a 5-mm-diameter circular area using a sterilized needle. A 10 µl aliquot of 1 × 106 conidia/ml suspension of each of the isolates (MG2-1 and MG3-1) was inoculated into each wound. For each isolate, there were six replicate fruits. The control fruits were inoculated with sterile distilled water. The same inoculation was applied to 12 non-wound papaya fruits. Fruits were then placed in boxes which were first washed with 75% alcohol and lined with autoclaved filter paper moistened with sterilized distilled water to maintain high humidity. The boxes were then sealed and incubated at 28°C. After 10 days, all the inoculated fruits showed symptoms, while the fruits that were mock inoculated were without symptoms. Koch's postulates were fulfilled by re-isolation of C. siamense from diseased fruits. To our knowledge, this is the first report of C. siamense causing anthracnose of papaya in China. This finding will enable better control of anthracnose disease caused by C. siamense on papaya.

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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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