First report of root rot of Schisandra chinensis caused by Fusarium acuminatum in Northeast China

Wuweizi [Schisandra chinensis（Turcz.）Baill.] is used for traditional medicine in northeastern China. In August of 2019, root rot of S. chinensis with an incidence of 30%-50% was observed in a commercial field located in Liaozhong city (41º29’57” N, 122º52’33” E) in the Liaoning province of China. The diseased plants were less vigorous, stunted, and had leaves that turned yellow to brown. Eventually, the whole plant wilted and died. The diseased roots were poorly developed with brown lesion and eventually they would rot. To determine the causal agent, symptomatic roots were collected, small pieces of root with typical lesions were surface sterilized in 2% NaOCl for 3 min, rinsed three times in distilled water, and then plated onto PDA medium. After incubation at 26°C for 5 days, whitish-pink or carmine to rose red colonies on PDA were transferred to carnation leaf agar (CLA). Single spores were isolated with an inoculation needle using a stereomicroscope. Five single conidia isolates obtained from the colonies were incubated at 26°C for 7 days, abundant macroconidia were formed in sporodochia. Macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, mostly 3 to 5 septate, measuring 31.3 to 47.8 × 4.8 to 7.5µm (n=50). Microconidia were oval and irregular ovals, 0-1 septate, measuring 5.0 to 17.5 × 2.5 to 17.5µm (n=50). Chlamydospores formed in chains on within or on top of the mycelium. Morphological characteristics of the isolates were in agreement with Fusarium acuminatum (Leslie and Summerell, 2006). To confirm the identity, the partial sequence of the translation elongation factor 1 alpha (TEF1-á) gene of five isolates was amplified using the primers EF-1(ATGGGTAAGGARGACAAG) and EF-2 (GGARGTACCAGTSATCATGTT) (O’Donnell et al. 2015 ) and sequenced. The rDNA internal transcribed spacer (ITS) region for the five isolates was also amplified using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTATTGATATGC) (White et al.1990) and sequenced. The identical sequences were obtained, and one representative sequence of isolate WW31-5 was submitted to GenBank. BLASTn analysis of the TEF-á sequence (MW423624) and ITS sequence (MZ145386), revealed 100%（708/685bp, 563/563bp）sequence identity to F. acuminatum MH595498 and MW560481, respectively. Pathogenicity tests were conducted in greenhouse. Inoculums of F. acuminatum was prepared from the culture of WW31-5 incubated in 2% mung beans juice on a shaker (140 rpm) at 26°C for 5 days. Ten roots of 2-years old plants of S. chinensis were immersed in the conidial suspension (2 × 105 conidia/ml) for 6 hours, and another ten roots immersed in sterilized distilled water in plastic bucket for 6 hours. All these plants were planted into pots with sterilized field soil (two plants per pot). Five pots planted with inoculated plants and another five pots planted with uninoculated plants served as controls. All ten pots were maintained in a greenhouse at 22-26°C for 21 days and irrigated with sterilized water. The leaves of the inoculated plants became yellow，gradually dried up, eventually finally all the aboveground parts died. The roots of the inoculated plants were rotted. Non-inoculated control plants had no symptoms. F. acuminatum was reisolated from the roots of inoculated plants and had morphology identical to the original isolate. The experiment was repeated twice with similar results. F. acuminatum has been reported as a pathogen caused root rot of ginseng (Wang et al. 2016) and not reported on Wuweizi in China. To our knowledge, this is the first report of root rot of S. chinensis caused by F. acuminatum. We have also observed the disease at Benxi city of Liaoning Province in 2020 and it has become an important disease in production of S. chinensis and the effective control method should be adopted to reduce losses.

Genetic diversity and potential inoculum sources of Fusarium species causing cankers in bareroot-propagated almond trees in California nurseries

Previous research determined that Fusarium acuminatum and Fusarium avenaceum are important causal agents of a canker disease in bareroot-propagated fruit and nut trees in California that emerges during cold-storage or after transplanting. The disease largely disappeared after 2001, but it reemerged in 2011 in almond trees in at least one nursery. This motivated further study of the etiology and epidemiology of the disease by undertaking studies to determine distribution of the pathogens throughout almond nursery propagation systems and trace possible sources of inoculum. Research initiated in 2013 detected pathogenic Fusarium spp. throughout the almond propagation system, including in healthy trees, in soils, on wheat rotation crops, on equipment, and in the cold storage facility air. In addition to the two Fusarium spp. implicated previously, Fusarium brachygibbosum and a new Fusarium species, Fusarium californicum, were found to be pathogenic on almond trees. Multi-locus sequence typing and somatic compatibility testing confirmed that isolates within a species collected from different materials in the nursery were all highly genetically similar and likely of one clonal lineage. These findings affirm that equipment surfaces, wheat rotation crops, soil, cold storage facility air, and asymptomatic almond tree materials (i.e., rootstock cuttings, budwood, and scions) can potentially contribute inoculum to increase disease prevalence and severity.

First report of root rot of Polygonatum odoratum caused by Fusarium acuminatum in China

Polygonatum odoratum (Mill.) Druce is used in traditional Chinese medicine and also consumed as a vegetable. In July of 2020, a root rot was observed on P. odoratum in a commercial field located in Benxi city (41º23’32” N, 124º04’27” E), Liaoning province of China. About 35% diseased plants in the field exhibited poor vigor, were stunted, and had yellow or brown leaves. Affected plants wilted and died. Roots of the plants were poorly developed, had brown lesions, and later rotted. To determine the causal agent, symptomatic roots with typical lesions were cut into small pieces, surface sterilized in 2% sodium hypochlorite (NaOCl) for 3 min, rinsed three times in sterile water, and plated onto PDA medium. After 5 days of incubation at 26°C, whitish-pink to red colonies growing from the root samples were observed and transferred to carnation leaf agar (CLA). Ten single conidia isolates obtained from the colonies on CLA were incubated at 26°C for 10 days. Abundant macroconidia were formed in sporodochia on CLA. Macroconidia were falcate, slender, distinctively curved in the bottom half of the apical cell, had 3 to 5 septa, and 33.1 - 46.3 × 5.0 - 7.2 μm (n=50). Chlamydospores formed in chains or single, measuring 13.8 to 14.5 μm in diameter. Microconidia were not observed on CLA. Morphologically, the isolates were identified as Fusarium acuminatum (Leslie and Summerell, 2006). To confirm the species identity, the partial translation elongation factor 1 alpha (TEF1-α) gene and rDNA internal transcribed spacer (ITS) region of isolate YZ5-2 were amplified and sequenced (O’Donnell et al. 2015; White et al.1990). BLASTn analysis of both TEF sequence (MW423623) and ITS sequence (MW423626), revealed 100% (696/692 bp) and 99.64% (563/602 bp) sequence identity with F. acuminatum LC546967 and MF509746, respectively. Pathogenicity tests were carried out in the greenhouse. A conidial suspension (2 × 106 conidia per ml) of the isolate YZ5-2 was prepared from 7-day-old cultures grown in potato dextrose broth (PDB) o n a shaker (140 rpm) at 26±1°C. Five 12-liter pots were filled with sterilized field soil and each pot was drenched with 300ml of conidial suspension. Five control pots with sterilized field soil and 300 ml PDB were also included. Roots of 20 healthy P. odoratum plants were surface disinfected in 2% NaOCl for 3 min, and rinsed with sterilized water. Prior to planting, 2-3 pinholes (1.5× 1.0 mm) were made using a toothpick on the root surface of each plant, and they were then planted in each pot (2 plants per pot). All ten pots were maintained in a greenhouse at 22-26°C for 40 days. Plants grown in the pots inoculated with the conidial suspension were stunted, had yellowed leaves and were wilted. The roots of the affected plants were rotted. Disease symptoms were similar to those observed in field. Non-inoculated control plants had no symptoms. F. acuminatum was reisolated from inoculated plants and was identical to the original isolate. The experiment was repeated twice with similar results. To our knowledge, this is the first report of root rot of P. odoratum caused by F. acuminatum in China. The disease has since been observed on P. odoratum in fields in Liaoyang and Qingyuan city in Liaoning Province of China, and it has become an important threat to P. odoratum production in China.

First Report of Post-Harvest Tuber Rot of American Groundnut (Apios americana) Caused by Fusarium acuminatum

Apios americana Medik, commonly known as American groundnut, is a leguminous perennial vine crop native to North America and is cultivated in Japan and Korea (Chu et al. 2019). Its tubers are edible and believed to be very nutritious, especially for women just after childbirth. The tubers also contain secondary metabolites, saponin and genistein, which is good for human health (Ichige et al. 2013). However, the storage of tubers at inappropriate temperatures and humidity levels can cause severe fungal infection, and adversely affect tuber quality. During March and April 2020, a white to pale-orange fungal mycelia were observed on stored American groundnut tubers, with 10 to 15% of seed tubers rotten. Infected tubers were collected, and fungal isolates were isolated on potato dextrose agar (PDA) using the single spore isolation method (Leslie and Summerell 2006). A pure culture (isolate JC20003) was obtained and stored at the Bioenergy Crop Research Institute, NICS, Muan, Republic of Korea. The fungus was cultured on PDA and V8 liquid media for 7 days at 25℃ to observe its morphological characteristics. The length and width of macroconidia ranged from 20.6 to 52.9 μm and 2.9 to 5.1 μm, respectively (n = 30). The microconidia were 8.5 to 14.9 μm and 2.3 to 4.2 μm in length and width, respectively (n = 30). Macroconidia were broadly falcate, strongly septate, 2 to 6 septations with dorsiventral curvature; chlamydospores were formed in chains; and microconidia were fusiform with 0 to 1 septation observed. Genomic DNA of the isolate was extracted using Solgent DNA extraction kit (Solgent, Daejeon, Korea), followed by PCR analysis using the internal transcribed spacer (ITS5/ITS4) and elongation factor (EF-1/EF2) genes (White et al. 1990; O’Donnel 2000). PCR products were sequenced and analyzed to confirm species identity (Yang et al. 2018). These sequences were deposited in GenBank (accession numbers MT703859/ITS and MT731939/EF). BLASTn search analysis showed 100% sequence similarity with Fusarium acuminatum (isolates N-51-1/ITS and WXWH24/EF). Based on morphological and molecular data analysis, the fungus was identified as F. acuminatum (Leslie and Summerell 2006; Marin et al. 2012). Pathogenicity tests were conducted on five tubers inoculated with 5 mm mycelial plugs with three replicates, while a non-mycelial plug served as the control. After 5 days of incubation in plastic containers at 25 °C with high humidity, typical symptoms developed. No symptoms were observed on the control tubers; F. acuminatum was re-isolated from artificially inoculated tubers to complete Koch’s postulates. This is the first report on post-harvest tuber rot caused by F. acuminatum in Apios americana.

Fusarium acuminatum Associated with Root Rot of Ophiopogon japonicus (Linn. f.) in China

Ophiopogon japonicus (Linn. f.) is a perennial evergreen in the Liliaceae family that is cultivated in many provinces of China due to its high medicinal and economic value . In April 2019, an unknown root rot disease was observed on the rhizomes of O. japonicus in a commercial production field in Xiangyang City (30.83° N, 112.53° E), Hubei Province. Disease incidence was approximately 10-20%. Symptoms included chlorosis, drooping and rolling of the leaves followed by rapid death of entire plant. Infected roots appeared to be softened, necrotic, and shriveled with reddish fungal growth. Infected tissues were disinfested on surface with 75% ethanol for 30 s and 0.1% HgCl2 for 1 min, rinsed with sterile distilled water, and dried. Small pieces (2 mm × 2 mm) were then excised from disinfested tissue and incubated on potato dextrose agar (PDA) medium at 25 ℃ in the dark. After 3 days of incubation, six isolates with 75% of isolation rate and same colony morphology were sub-cultured and purified by hyphal tip isolation. Purified cultures grew rapidly and media plates (70×70 mm ) were covered with hyphae after 3 to 4 days. Cultures were initially white and became pink or red over 5 days. Microconidia were not observed. Macroconidia were produced from monophialides on branched conidiophores, which were slender, equilaterally curved, and measured 32.5 to 53.5 μm in length and 3.5 to 5.1 μm in width, with three to five septa. All strains were preliminarily identified as Fusarium acuminatum (Eslie and Summerell 2006) on the basis of morphology. To confirm the identity of the pathogen, molecular identification was performed with strain MD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were internal transcribed spacer (ITS), RNA polymerase second largest subunit (RPB2) and beta-tubulin gene (TUB2) regions of rDNA, using ITS1,4 (Yin et al. 1990) , RPB2-5f2/7cr (O’Donnell et al. 2010)and Btu-F-F01, Btu-F-R01 primers(Wang et al. 2014), respectively. Nucleotide sequences were deposited in NCBI (GenBank MT525360.1; MW164629; MT588110.1). BLAST analysis of the ITS sequence had 100% similarity to a 517 bp portion of F. acuminatum sequence in GenBank (MK764994.1) ；RPB2 sequence had 100% similarity to a 687 bp portion of F. acuminatum sequence in GenBank (HM068330.1) and TUB2 sequence had 99% similarity to a 964 bp portion of F. acuminatum sequence in GenBank (KT965741.1). A pathogenicity test was performed in laboratory on O. japonicus roots with isolate MD1. Mycelial plugs (5 mm) were excised from the margin of colony cultured for 5 days, and placed on three-years-old tuberous roots covered with wet sterile cotton and kept at 25℃, under 80% relative humidity. Controls were inoculated with non-colonized PDA plugs (5 mm). All treatments had three replicate plants. On incolated plants, white hyphae covered on O. japonicus roots 3 DPI became pink and by 5 DPI, roots had rot symptoms. By comparision, the control plants had no symptoms. The pathogen was reisolated from the inoculated roots and exhibited same morphological characteristics and ITS sequence as those of F. acuminatum. F. acuminatum was reported to cause fruit rot on postharvest pumpkin and Vaccinium corymbosum in China (Li et al. 2019; Wang et al. 2016).To our knowledge, this is the first report of root rot caused by F. acuminatum on O. japonicus in China.

Cross Pathogenicity Studies Show South Dakota Isolates of Fusarium acuminatum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. solani, and F. subglutinans from Either Soybean or Corn are Pathogenic to Both Crops

In South Dakota, despite that integrated pest management options are available, Fusarium root rot is an emerging disease on soybean (Glycine max L.) and corn (Zea mays L.). Surveys were conducted across South Dakota on soybean and corn fields in 2014 and 2015, respectively, to assess the prevalence of species of Fusarium causing root rot. Fusarium acuminatum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. solani, and F. subglutinans were identified common to soybean and corn. A total of 21 isolates, representing these seven species, were evaluated for their pathogenicity on soybean (‘Williams 82’) and corn (‘B73’) using the inoculum layer inoculation method in the greenhouse. At 14 days postinoculation, the seedlings were evaluated for root rot severity (1-to-5 rating scale), and relative treatment effects (RTEs) were estimated. A significant effect of the treatments was observed on RTE for soybean (P = 1.1 × 10−7) and corn (P = 3.0 × 10−14). Two F. proliferatum isolates and one F. graminearum isolate from corn caused significantly greater RTE than the other treatments (including the noninoculated control) on soybean and corn. Results indicate that soybean and corn can serve as inoculum sources of the seven species of Fusarium that are pathogenic to both crops.