scholarly journals First Report of Fusarium culmorum Causing Maize Stalk Rot in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Laikun Xia ◽  
Yanyong Cao ◽  
Jie Wang ◽  
Jie Zhang ◽  
Shengbo Han ◽  
...  

Maize stalk rot has become one of the most important diseases in maize production in China. From 2017 to 2019, a survey was conducted to determine the population diversity of Fusarium species associated with maize diseases in 18 cities across Henan Province. Maize stalk rot with an incidence of more than 20% that caused yield losses up to 30% was observed on maize variety Zhengdan958, which was grown in two continuous maize fields in Zhumadian City, Henan Province. The stem tissues from the boundary between diseased and healthy pith were chopped into small pieces (3 × 8 mm), disinfected (70% ethanol for 1 min) and then placed onto potato dextrose agar (PDA) amended with L-(+)-Lactic-acid (1 g/L) and incubated at 25°C for 4 days. Colonies on PDA produced fluffy, light yellow aerial mycelium and purple to deep brick red pigment at 25°C (Fig 1A, 1B). On carnation leaf agar (CLA), macroconidia in orange sporodochia formed abundantly, but microconidia were absent. Macroconidia were short and thick-walled, had 3 to 5 septa, a poorly developed foot cell and rounded apical cell (Fig 1C). These characteristics matched the description of Fusarium culmorum (Leslie and Summerell 2006) and isolates DMA268-1-2 and HNZMD-12-7 were selected for further identity confirmation. Species identification was confirmed by partial sequences of three phylogenic loci (EF1-α, RPB1, and RPB2) using the primer pairs EF1/EF2, CULR1F/CULR1R, and CULR2F/CULR2R, respectively (O'Donnell et al., 1998). The consensus sequences from the two isolates were deposited in GenBank (MZ265416 and MZ265417 for TEF, respectively; MZ265412 and MZ265414 for RPB1, respectively; MZ265413 and MZ265415 for RPB2). BLASTn searches indicated that the nucleotide sequences of the three loci of the two isolates revealed 99% to 100% similarity to those of F. culmorum strains deposited in the GenBank, Fusarium-ID, and MLST databases (Supplementary Table 1~3). Pathogenicity test was conducted at the flowering-stage using Zhengdan958 and Xundan20 plants according to previously described method (Zhang et al., 2016; Cao et al., 2021; Zhang et al., 2021). The second or third internodes of thirty flowering plants were drilled to make a wound approximately 8 mm in diameter using an electric drill. Approximately 0.5 mL inoculum (125 mL colonized PDA homogenized with 75 mL sterilized distilled water) was injected into the wound and sealed with Vaseline and Parafilm to maintain moisture and avoid contamination. Sterile PDA slurry was used as a control. Thirty days after inoculation, the dark-brown, soft rot of pith tissues above and below the injection sites were observed, and some plants were severely rotten and lodged (Fig 1D, 1E). These symptoms were similar to those observed in the field. No symptoms were observed on control plants. The same pathogen was re-isolated from the inoculated stalk lesions but not from the control, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of F. culmorum as the causal agent of stalk rot on maize plants in China. Also, this fungus has been reported to cause maize ear rot in China (Duan et al. 2016) and produce mycotoxins such as trichothecenes, nivalenol, and zearalenone that cause toxicosis in animals (Leslie and Summerell 2006). The occurrence of maize stalk rot and ear rot caused by F. culmorum should be monitored due to the potential risk for crop loss and mycotoxin contamination.

Plant Disease ◽  
2021 ◽  
Author(s):  
Shengbo Han ◽  
Yanyong Cao ◽  
Jie Zhang ◽  
Jie Wang ◽  
Lili Zhang ◽  
...  

In a field survey from 2017 to 2019, Fusarium stalk rot symptoms including discolored, disintegrated stalk pith tissues and lodged plants were observed in maize hybrid lines Fuyu1611, Jidan66, and Danyu8439 grown in fields in Anshan (40o49′39′′N, 122 o34′6′′E), Liaoning province. Its incidence ranged from 15% to 20% and caused a yield loss of up to 30%. Infected pieces of stem tissues were dissected and then sterilized with 1% NaOCl for 1 min, 70% ethanol for 1 min, rinsed 3 times with sterilized ddH2O, and dried with filter paper in hood. Three pieces were placed onto Potato dextrose agar (PDA) and incubated at 25 °C for 5 days. The colonies were single-spore subcultured on PDA at 25 °C for 2 weeks (Leslie and Summerell 2006). Morphological features were observed on PDA and carnation leaf agar (CLA). The average mycelial growth rate was 4.5 to 10.3 mm/day at 25 °C on PDA. The colonies produced aerial mycelia, varying from dense white to grayish-rose, and secreted red pigments in the agar (Fig. 1A; 1B). Macroconidia produced on CLA were long and relatively slender, commonly 4- to 7-septate, averaging 85.6 × 5.2 μm, with thick walls and pronounced dorsiventral curvature with a distinctly foot-shaped and elongated basal cell and an apical cell that was whip-like (Fig. 1C). Microconidia were rarely observed on PDA or CLA. Morphological characteristics of the isolates were similar to the features of Fusarium longipes as previously described (Leslie and Summerell 2006). The portions of three phylogenic loci (EF1-α, RPB1, RPB2) were PCR amplified using the primer pairs EF1/EF2 (O'Donnell et al, 1998), lonR1F/lonR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3 and 5-CCAATGGACTGGGCAGCCAAAACGCC-3) and lonR2F/lonR2R (5-TATACATTTGCCTCCACTCTTTCCCAT-3 and 5-CGGAGCTTGCGTCCGGTGTGGCCGTTG-3) and sequenced. The consensus sequences were submitted to GenBank (MT513215 and MT997083 for TEF, MT513213 and MT997088 for RPB1; MT513214 and MW020572 for RPB2). BLASTn searches indicated that the nucleotide sequences of the three loci of the two isolates shared 94.52% to 99.69% identity with sequences of F. longipes strains deposited in the GenBank, Fusarium-ID and Fusarium MLST databases (Supplementary Table 1, 3, 4). A phylogram inferred via maximum likelihood analysis of the combined EF-1α, RPB1, RPB2 partial sequence data of Fusarium species (Supplementary Table 2) was inferred using the CIPRIES website (https://www.phylo.org). Isolates LNAS-05-A and LNAS-09-A clustered with F. longipes, with 98% bootstrap support (Fig. 2). Pathogenicity tests were conducted on three-leaf-stage seedlings and flowering-stage c.v. Zhengdan958 and B104 plants according to previously described methods (Ye et al., 2013; Zhang et al. 2016) with minor modifications. Three days after the roots of the seedlings were inoculated with 1 × 106 macroconidia solution, the leaves and stems exhibited typical wilt symptoms (Fig. 1D). Twenty flowering-stage maize plants were drilled individually at the second or third node above the soil using an electric drill (Bosch TSR1080-2-Li) to create a hole (8 mm in diameter). An approximately 0.5 mL mycelia plug (125 mL homogenized hyphal mats + 75 mL sterilized ddH2O) was injected into the hole and covered with Vaseline. Sterilized PDA plugs were used as a control. The stalk tissue of the split internodes turned dark brown and the brown area expanded above and below the injection site by 14 dpi. All of the inoculated plants developed characteristic stalk rot symptoms, whereas no symptoms were observed in the controls (Fig. 1E). The pathogen was re-isolated, and its identity was confirmed by sequencing the above mentioned loci. F. longipes was generally regarded as a tropical Fusarium species (Leslie and Summerell 2006). This is the first report that F. cf. longipes can cause stalk rot of maize under filed condition in a temperate, typical corn belt region of China.


Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1156-1156 ◽  
Author(s):  
H. Zhang ◽  
W. Luo ◽  
Y. Pan ◽  
J. Xu ◽  
J. S. Xu ◽  
...  

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.


Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 496-496 ◽  
Author(s):  
A. Garibaldi ◽  
G. Gilardi ◽  
A. Poli ◽  
M. L. Gullino

In the summer of 2009, a wilt of chicory was observed on 25 to 30% of 30-day-old Cichorium intybus L. cv. Clio plants grown outdoors on a commercial farm in Piedmont (northern Italy). Affected plants were chlorotic and stunted with poorly developed root systems compared with healthy plants. Black streaks were observed in the stem and proximal part of the leaf vascular system in wilted plants. Fusarium oxysporum Schltdl. was isolated from symptomatic vascular tissue on a Fusarium-selective medium (1) from 80% of samples. Grown on potato dextrose agar (PDA) for 4 days at 23°C, the colonies, initially white and later pale pink, produced hyaline microconidia that were oval-elliptical and cylindrical in shape measuring 5.6 to 14.9 (average 10.2) × 2.1 to 4.5 (3.0) μm, borne on short monophialides measuring 8.2 to 16.1 (average 13.2) × 2.1 to 4.2 (3.3) μm. Macroconidia were slightly curved, three-septate, with a slightly hooked apical cell and a foot-shaped basal cell measuring 24.9 to 41.6 (average 32.2) × 3.2 to 5.2 (4.3) μm. Chlamydospores were both terminally and intercalary, solitary but also in short chains (2 to 4 elements) measuring 21.1 to 41.0 (average 27.2) μm (2). The internal transcribed spacer (ITS) rDNA region was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 527-bp amplicon (GenBank Accession No. HQ644423) obtained had 98% sequence identity with F. oxysporum (GenBank Accession No. FJ605247). The translation elongation factor-1α (EF-1α) gene was amplified using primers EF-1/EF-2 and sequenced (GenBank Accession No. GU564259). The 663-bp fragment had 99% sequence identity with F. oxysporum (GenBank Accession Nos. EU313540, EU313539, and DQ837696). Pathogenicity tests were conducted on 15-day-old chicory plants from two cultivars (Clio and Katia). Thirty-five plants per cultivar were inoculated by dipping their roots in a 1 × 106 CFU/ml suspension of isolate FusCic45B recovered from wilted chicory. Inoculated and noninoculated plants were transplanted into five pots filled with 10 liters of steamed mix (peat/perlite/sand, 60:20:20 vol/vol) and were maintained in a glasshouse at 25 to 27°C. Wilt symptoms and vascular discoloration of the roots, crown, and veins developed 15 days after inoculation on all inoculated plants. Plants of cv. Clio were more susceptible. F. oxysporum was always reisolated from infected plants using the Fusarium-selective medium. All noninoculated plants remained healthy. The pathogenicity test was conducted twice. To our knowledge, this is the first report of wilt caused by F. oxysporum on chicory, C. intybus, in Italy as well as worldwide. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983.


Plant Disease ◽  
2010 ◽  
Vol 94 (8) ◽  
pp. 1069-1069 ◽  
Author(s):  
J. C. Bienapfl ◽  
D. K. Malvick ◽  
J. A. Percich

Multiple Fusarium species have been found in association with soybean (Glycine max) plants exhibiting root rot in the United States (3). Soybean plants that lacked apparent foliar symptoms, but exhibited 2- to 5-mm brown, necrotic taproot lesions and lateral root necrosis were observed in Minnesota in one field each in Marshall and Otter Tail counties in July of 2007, as well as in one field in Marshall County in July of 2008. Sampling was conducted as part of a study investigating root rot in major soybean-production areas of Minnesota. Plants were arbitrarily dug up at the R3 growth stage. Root systems were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, and dried. Fusarium isolates were recovered from root sections with necrotic lesions embedded in modified Nash-Snyder medium (1). One resulting Fusarium colony from one plant per county was transferred to half-strength acidified potato dextrose agar (PDA) and carnation leaf agar (CLA) to examine morphological characteristics (4). Culture morphology on PDA consisted of flat mycelium with sparse white aerial mycelium. On CLA, thick-walled macroconidia with a hooked apical cell and a foot-shaped basal cell were produced in cream-colored sporodochia. Macroconidia ranged from 32.5 to 45.0 μm long. Microconidia were oval to cylindrical with 0 to 1 septa, ranged from 7.5 to 11.25 μm long, and were produced on monophialides. Chlamydospores were produced abundantly in chains that were terminal and intercalary in the hyphae of 4-week-old cultures. Morphological characteristics of the three isolates were consistent with descriptions of F. redolens (2,4). The identity of each isolate was confirmed by sequencing the translation elongation factor 1-α (TEF) locus (4). BLAST analysis of the TEF sequences from each isolate against the FUSARIUM-ID database resulted in a 100% match for 17 accessions of F. redolens (e.g., FD 01103, FD 01369). Each F. redolens isolate was tested for pathogenicity on soybean. Sterile sorghum grain was infested with each isolate and incubated for 2 weeks. Sterile sorghum was used for control plants. Soybean seeds of cv. AG2107 were planted in 11.4-cm pots ~1 cm above a 25-cm3 layer of infested sorghum or sterile sorghum. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting. Each F. redolens isolate consistently caused taproot necrosis on inoculated plants, whereas control plants did not exhibit root necrosis. Isolations were made from roots of inoculated and control plants and the isolates recovered from inoculated plants were identified as F. redolens based on morphological characteristics and TEF sequences. Fusarium species were not isolated from control plants. To our knowledge, this is the first report of F. redolens causing root rot of soybean; however, it is possible F. redolens has been found previously and misidentified as F. oxysporum (2,4). Results from inoculations suggest that F. redolens may be an important root rot pathogen in Minnesota soybean fields. References: (1) J. C. Bienapfl et al. Acta Hortic. 668:123, 2004. (2) C. Booth and J. M. Waterston. No. 27 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1964. (3) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.


Plant Disease ◽  
2011 ◽  
Vol 95 (9) ◽  
pp. 1191-1191 ◽  
Author(s):  
A. Garibaldi ◽  
D. Bertetti ◽  
P. Pensa ◽  
A. Poli ◽  
M. L. Gullino

During summer 2010, symptoms of a wilt disease were observed in a commercial farm in northern Italy on Crassula ovata (jade plant). First symptoms consisted of chlorosis and premature drop of still turgid leaves. As the disease progressed, leaves turned yellow and wilted before dropping off and the stem wilted, bent, and eventually rotted starting from the base. In some cases, the stem broke or the basal portion of the leaf rotted. Brown discolorations were observed in the vascular system. Of 10,000 plants, 65% (cv. Mini) and 5% of 600 plants (cv. Magical Tree) were affected. Premature dropping of leaves was more frequent on cv. Magical Tree. Using the Komada's Fusarium-selective medium, a fungus was consistently and readily isolated from symptomatic vascular tissues of plants belonging to both cultivars. Isolates obtained from both cultivars were purified, subcultured on potato dextrose agar (PDA), and single-spore cultures were obtained. On PDA, both isolates produced pale violet, abundant, aerial mycelium, felted in old cultures, with purple pigments in the agar. The isolates were grown on Spezieller Nährstoffarmer agar for characterization of macroconidia and microconidia (1). Both isolates produced sparse, 3 to 5 septate, nearly straight macroconidia measuring 30 to 56 × 3 to 5 (average 40 × 4) μm with a short apical cell and a foot-shaped basal cell. Sporodochia were not observed. Unicellular, oval-elliptical microconidia measuring 5 to 13 × 3 to 4 (average 8 × 3) μm were produced on short monophialides. Chlamydospores were abundant, single and sometime in pairs, terminal and intercalary, rough walled, and measured 6 to 9 μm. Such characteristics are typical of Fusarium oxysporum (3). The ITS region (internal transcribed spacer) of rDNA was amplified with primers ITS1/ITS4 (4) and sequenced. BLASTn analysis of an isolate from C. ovata cv. Mini (515 bp, Accession No. HQ682196) and C. ovata cv. Magical Tree (509 bp, Accession No. HQ682197) showed an E-value of 0.0 with F. oxysporum. For these sequences, pairwise alignment of EMBOSS (E.B.I. - The European Bioinformatics Institute) revealed identity and similarity of 99.0%. To confirm pathogenicity, tests were conducted on 5-month-old plants of cvs. Mini and Magical Tree. Plants (three per treatment) were inoculated by dipping roots in a 1 × 106 CFU/ml conidial suspension of the two isolates of F. oxysporum prepared from 10-day-old cultures grown on casein liquid medium (2), shaken (90 rpm) for 10 days at 24°C ± 1 (12-h fluorescent light, 12-h dark). Inoculated plants were transplanted into pots filled with steamed mix (sphagnum peat/perlite/pine bark/clay; 50:20:20:10) and maintained in a plant growth chamber at 25 ± 1°C under a regimen of 12 h per day of fluorescent light. Inoculated plants belonging to both cultivars showed typical first symptoms of Fusarium wilt after 13 days. In the following days, leaves dropped, stems wilted, and plants died. Noninoculated plants remained healthy. F. oxysporum was reisolated from inoculated plants. The pathogenicity test was conducted twice. This is, to our knowledge, the first report of F. oxysporum on C. ovata in Italy or worldwide. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Professional, Ames, IA, 2006. (2) A. Minuto et al. Phytoparasitica 36:294, 2008. (3) B. A. Summerell et al. Plant Dis. 87:117, 2003. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.


Plant Disease ◽  
2021 ◽  
Author(s):  
Xianping Zhang ◽  
Jiwen Xia ◽  
Jiakui Liu ◽  
Dan Zhao ◽  
Lingguang Kong ◽  
...  

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelons; among them, Fusarium spp. is the most important pathogen, affecting fruit yield and quality (Wang et al. 2011). In May 2017, fruit rot symptoms were observed on ripening muskmelons (cv. Jipin Zaoxue) in several fields in Liaocheng of Shandong Province, China. Symptoms appeared as brown, water-soaked lesions, irregularly circular in shape, with the lesion size ranging from a small spot (1 to 2 cm) to the decay of the entire fruit. The core and the surface of the infected fruit were covered with white to rose-reddish mycelium. Two infected muskmelons were collected from each of two fields, 10 km apart. Tissues from the inside of the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25 °C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), macroconidia had a pronounced dorsiventral curvature, falcate, 3 to 5 septa, with tapered apical cell, and foot-shaped basal cell, measuring 19 to 36 × 4 to 6 μm. Chlamydospores were abundant, 5.5–7.5 μm wide, and 5.5–10.5 μm long, ellipsoidal or subglobose. No microconidia were observed. These morphological characteristics were consistent with the descriptions of F. pernambucanum (Santos et al. 2019). Because these isolates had similar morphology, one representative isolate was selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolate using the CTAB method. The nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), translation elongation factor 1-α gene (TEF1), RNA polymerase II second largest subunit gene (RPB2), calmodulin (CAM) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (MN822926, MN856619, MN856620, and MN865126). Based on the combined dataset of ITS, TEF1, RPB2, CAM, alignments were made using MAFFT v. 7, and phylogenetic analyses were processed in MEGA v. 7.0 using the maximum likelihood method. The studied isolate (XP1) clustered together with F. pernambucanum reference strain URM 7559 (99% bootstrap). To perform pathogenicity test, 10 μl of spore suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25 °C, the interior of the inoculated muskmelons begun to rot, and the rot lesion was expanded from the core towards the surface of the fruit, then white mycelium produced on the surface. The same fungus was re-isolated from the infected tissues and confirmed to fulfill the Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of F. pernambucanum causing of fruit rot of muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.


Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 420-420 ◽  
Author(s):  
S. Chebil ◽  
R. Fersi ◽  
A. Yakoub ◽  
S. Chenenaoui ◽  
M. Chattaoui ◽  
...  

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 μm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 μm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 μm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia. References: (1) M. Chattaoui et al. Plant Dis. 96:905, 2012. (2) H. S. Fawcett. Calif. Citrogr. 16:208, 1931. (3) B. T. Linaldeddu et al. J. Plant Pathol. 91:234. 2009. (4) B. T. Linaldeddu et al. Phytopathol. Mediterr. 47:258, 2008.


Plant Disease ◽  
2014 ◽  
Vol 98 (2) ◽  
pp. 279-279 ◽  
Author(s):  
J.-H. Wang ◽  
H.-P. Li ◽  
J.-B. Zhang ◽  
B.-T. Wang ◽  
Y.-C. Liao

From September 2009 to October 2012, surveys to determine population structure of Fusarium species on maize were conducted in 22 provinces in China, where the disease incidence ranged from 5 to 20% in individual fields. Maize ears with clear symptoms of Fusarium ear rot (with a white to pink- or salmon-colored mold at the ear tip) were collected from fields. Symptomatic kernels were surface-sterilized (1 min in 0.1% HgCl2, and 30 s in 70% ethanol, followed by three rinses with sterile distilled water), dried, and placed on PDA. After incubation for 3 to 5 days at 28°C in the dark, fungal colonies displaying morphological characteristics of Fusarium spp. (2) were purified by transferring single spores and identified to species level by morphological characteristics (2), and DNA sequence analysis of translation elongation factor-1α (TEF) and β-tubulin genes. A large number of Fusarium species (mainly F. graminearum species complex, F. verticillioides, and F. proliferatum) were identified. These Fusarium species are the main causal agents of maize ear rot (2). Morphological characteristics of six strains from Anhui, Hubei, and Yunnan provinces were found to be identical to those of F. kyushuense (1), which was mixed with other Fusarium species in the natural infection in the field. Colonies grew fast on PDA with reddish-white and floccose mycelia. The average growth rate was 7 to 9 mm per day at 25°C in the dark. Reverse pigmentation was deep red. Microconidia were obovate, ellipsoidal to clavate, and 5.4 to 13.6 (average 8.8) μm in length. Macroconidia were straight or slightly curved, 3- to 5-septate, with a curved and acute apical cell, and 26.0 to 50.3 (average 38.7) μm in length. No chlamydospores were observed. Identity of the fungus was further investigated by sequence comparison of the partial TEF gene (primers EF1/2) and β-tubulin gene (primers T1/22) of one isolate (3). BLASTn analysis of the TEF amplicon (KC964133) and β-tubulin gene (KC964152) obtained with cognate sequences available in GenBank database revealed 99.3 and 99.8% sequence identity, respectively, to F. kyushuense. Pathogenicity tests were conducted twice by injecting 2 ml of a prepared spore suspension (5 × 105 spores/ml) into maize ears (10 per isolate of cv. Zhengdan958) through silk channel 4 days post-silk emergence under field conditions in Wuhan, China. Control plants were inoculated with sterile distilled water. The ears were harvested and evaluated 30 days post-inoculation. Reddish-white mold was observed on inoculated ears and the infected kernels were brown. No symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the pathogen from infected kernels. F. kyushuense, first described on wheat in Japan (1), has also been isolated from rice seeds in China (4). It was reported to produce both Type A and Type B trichothecene mycotoxins (1), which cause toxicosis in animals. To our knowledge, this is the first report of F. kyushuense causing maize ear rot in China and this disease could represent a serious risk of yield losses and mycotoxin contamination in maize and other crops. The disease must be considered in existing disease management practices. References: (1) T. Aoki and K. O'Donnell. Mycoscience 39:1, 1998. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) F. Van Hove et al. Mycologia 103:570, 2011. (4) Z. H. Zhao and G. Z. Lu. Mycotaxon 102:119, 2007.


Plant Disease ◽  
2022 ◽  
Author(s):  
Martina Sanna ◽  
Massimo Pugliese ◽  
Maria Lodovica GULLINO ◽  
Monica Mezzalama

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.


Plant Disease ◽  
2022 ◽  
Author(s):  
Xianping Zhang ◽  
Xuedong Cao ◽  
Qingqing Dang ◽  
Yongguang Liu ◽  
Xiaoping Zhu ◽  
...  

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelon fruit, including Fusarium spp.. Fusarium spp. are the most important pathogen, affecting muskmelon fruit yield and quality (Wang et al. 2011). In August 2020, fruit rot symptoms were observed on ripening muskmelons (cv. Tianbao) in several fields in Jiyang District, Jinan City of Shandong Province, China. The incidences of infected muskmelon ranged from 15% to 30% and caused an average 20% yield loss. Symptoms appeared as pale brown, water-soaked lesions that were irregular in shape, with the lesion sizes ranging from a small spot (1 to 2 cm) to decay of the entire fruit. The core and surface of infected fruit were colonized and covered with white mycelia. Two infected muskmelons were collected from two fields, 3.5 km apart. Tissues removed from inside the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), 3 to 5 septate, falcate, with a pronounced dorsiventral curvature macroconidia with tapered apical cell, and foot-shaped basal cell, measuring 20 to 40 × 3.5 to 4.5 μm. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of F. luffae (Wang et al., 2019). Because these isolates had similar morphology, two representative isolates (XP11 and XP12) were selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolates using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (ITS: MW391509 and MW391510, CAM: MW392789 and MW392790, RPB2: MW392797 and MW392798, TEF1: MW392793 and MW392794). Alignments of a combined dataset of ITS, CAM, RPB2 and TEF1 were made using MAFFT v. 7, and phylogenetic analyses were conducted in MEGA v. 7.0 using the maximum likelihood method. The muskmelon isolates (XP11 and XP12) clustered together with the F. luffae reference strain LC12167 (99% bootstrap). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25°C, the interior of the inoculated muskmelons begun to rot, and the rot lesion expanded from the core towards the surface of the fruit, then white mycelia were produced on the surface. Ten isolations were re-isolated from the infected tissues and confirmed to fulfill Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of fruit rot caused by F. luffae in muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.


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