Sterilized Field Soil Research Articles

First Report of Didymella pomorum Causing Leaf Blight on Angelica sinensis in China.

Angelica sinensis (Oliv.) Diels, is a perennial herbaceous plant of the Umbelliferae family. It has a long history of cultivation and is highly valued as a traditional Chinese medicine in China (Zhang et al. 2012). In September 2023, leaf blight on A. sinensis with an average disease incidence of 56% was recorded in an approximately 6.7-ha production field in Lijiang, Yunnan province, China (26.8215°N, 100.2369°E). At first, small, chlorotic lesions appeared on the leaves. They subsequently increased in density and gradually merged, causing leaves to yellow and wither. Ultimately the blight casused death of the entire foliage. In order to identify the causal agent, cross-sectional segments (5×5 mm2) were cut from the edge of leaf lesions, surface disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed three times with sterile distilled water. They were subsequently placed on potato dextrose agar (PDA) plates and incubated for 3 days under a 12-h photoperiod at 28℃. A total of ten isolates with similar morphological characteristics were obtained by single spore isolation. After 10 days of incubation on PDA, the colony morphology of these isolates was characterized by a brownish central area with a white edge. Aged colonies became wrinkled in the center of the colony. Conidia (n = 30) were elliptical and brown, with a size range of 4.11 to 6.55 μm (average 5.37±0.74 μm) × 3.17 to 4.62 μm (average 3.92±0.43 μm). Chlamydospores (n = 30) formed chains in series, spherical or elliptical in shape, ranging from yellow-brown to dark brown, with a size range of 12.30 to 13.70 μm (average 12.98±0.46 μm) × 4.20 to 5.30 μm (average 4.63±0.26 μm). The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of RNA polymerase II (RPB2), and the 28S nuclear ribosomal large subunit rRNA (LSU) region of two isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and LR0R/LR5 (Schoch et al. 2012), respectively. These amplicons were sequenced bidirectionally and assembled. The two isolates produced the same nucleotide sequences, and the sequences of a representative isolate (AsDp1) were deposited in GenBank. BLASTn analyses showed that the ITS (PP510616), RPB2 (PP526010), and LSU (PP550143) sequences of isolate AsDp1 were 100%, 99.66%, and 100% identical with those of Didymella pomorum ex-type isolate CBS 354.52 (MH857081, KT389616, and MH868616), respectively. A phylogenetic tree was constructed based on the ITS, RPB2, and LSU concatenated nucleotide sequences using the maximum likelihood method in MEGAX. Isolate AsDp1 was clustered with four D. pomorum isolates. According to the morphological and nucleotide sequences analyses, isolate AsDp1 was identified as D. pomorum (Chen et al. 2015). To determine pathogenicity, 1-year-old A. sinensis plants (approximately 20 cm tall) grown in 7-liter pots filled with sterilized field soil were sprayed until runoff with a 1×106 conidia/ml suspension of isolate AsDp1 onto the foliage, while control plants were sprayed with sterile water. All plants were cultivated under a 12-h photoperiod at 25℃. The pathogenicity tests were performed in triplicate with ten plants in each treatment. After fifteen days, numerous chlorotic lesions appeared on the leaves of all inoculated plants. The symptoms were similar to those found on naturally infected plants in the field, while the control plants remained asymptomatic. Subsequently, D. pomorum was reisolated from the diseased leaves, and the identity was confirmed based on its ITS sequence and morphological characteristics. D. pomorum causing stem canker on Rosa spp. was reported in Canada (Ilyukhin 2022). To our knowledge, this is the first report of D. pomorum causing leaf blight on A. sinensis in China. This etiological finding will potentially pave the way for the development of control strategies of this disease.

Trichoderma virens and Pseudomonas chlororaphis Differentially Regulate Maize Resistance to Anthracnose Leaf Blight and Insect Herbivores When Grown in Sterile versus Non-Sterile Soils.

Soil-borne Trichoderma spp. have been extensively studied for their biocontrol activities against pathogens and growth promotion ability in plants. However, the beneficial effect of Trichoderma on inducing resistance against insect herbivores has been underexplored. Among diverse Trichoderma species, consistent with previous reports, we showed that root colonization by T. virens triggered induced systemic resistance (ISR) to the leaf-infecting hemibiotrophic fungal pathogens Colletotrichum graminicola. Whether T. virens induces ISR to insect pests has not been tested before. In this study, we investigated whether T. virens affects jasmonic acid (JA) biosynthesis and defense against fall armyworm (FAW) and western corn rootworm (WCR). Unexpectedly, the results showed that T. virens colonization of maize seedlings grown in autoclaved soil suppressed wound-induced production of JA, resulting in reduced resistance to FAW. Similarly, the bacterial endophyte Pseudomonas chlororaphis 30-84 was found to suppress systemic resistance to FAW due to reduced JA. Further comparative analyses of the systemic effects of these endophytes when applied in sterile or non-sterile field soil showed that both T. virens and P. chlororaphis 30-84 triggered ISR against C. graminicola in both soil conditions, but only suppressed JA production and resistance to FAW in sterile soil, while no significant impact was observed when applied in non-sterile soil. In contrast to the effect on FAW defense, T. virens colonization of maize roots suppressed WCR larvae survival and weight gain. This is the first report suggesting the potential role of T. virens as a biocontrol agent against WCR.

First Report of Fusarium tricinctum Causing Root Rot on Chinese dwarf cherry (Cerasus humilis) in China.

Chinese dwarf cherry (Cerasus humilis) is a perennial small shrub indigenous to northern China, highly regarded for its calcium-rich fruit known as 'calcium fruit'. These fruits have a remarkable capacity to aid in human calcium absorption. Additionally, they contain beneficial flavonoids that hold promise for applications in the healthcare industry (Wang et al., 2018). In July 2020, a concerning development occurred on the farms in Jingtai County (37.48o N, 103.82o E), Baiyin City, Gansu Province in China. Approximately 20 to 30% of C. humilis at the full culture stage exhibited symptoms of root rot, including brownish leaves, rotten roots, and plant mortality, leading to a decrease of over 30% in calcium fruit yield. To identify the pathogen, the surface of symptomatic roots was sterilized with 3% NaOCl for 2 minutes, followed by 70% ethanol for 30 seconds, and rinsed three times with sterile water. Tissue sections (5×5mm) from the margin of the necrotic lesion were cut and cultured on potato dextrose agar (PDA) medium, and incubated for 7 days at 25℃. Five pure culture isolates were obtained from individual spores. Initially, the isolates exhibited abundant white aerial mycelia that turned light pink on the third day. Macroconidia were falciform, two to five septate, straight or slightly curved, and measuring 20.1 to 32.5×2.2 to 3.8 μm (n=50). Napiform microconidia were oval-ellipsoid, non-septate, and measuring 6.2 to 9.3×4.2 to 5.8 μm (n=50). Based on these morphological characteristics, the fungus was tentatively identified as Fusarium species (Leslie and Summerell, 2006). To confirm the identification, the internal transcribed spacer region (ITS) and the translation elongation factor (EF1α) of the isolate CH-2 were partially amplified and sequenced using the primers ITS1/ITS4 and EF2T/EF3 (Li et al., 2013, Yang et al., 2022). Upon comparison with the sequences in GenBank, 857 bp ITS sequence showed 100% homology to Fusarium tricinctum isolate QY3-1 (GenBank accessions no. MZ572963.1), and 665 bp EF1α sequence showed 99.7% homology to F. tricinctum strain TQC-C2 (GenBank accessions no. KF939493.1). The resulting sequences were deposited into GenBank with accession nos. OQ581576 and OQ848462 respectively. A maximum likelihood (ML) phylogenetic analysis based on combined partial ITS and EF1α data set was conducted via ML bootstrapping using MEGA 11. According to morphology and phylogenetic analysis, the isolate was identified as F. tricinctum (Wang et al., 2022). For a pathogenicity test, a pot experiment was conducted in a greenhouse with a temperature range of 20-27℃ and 60% relative humidity. Roots of C. humilis were immersed in a spore suspension (1×107 conidia/ml) of isolate F. tricinctum CH-2 for approximately 5 minutes. Subsequently the treated roots were planted in pots filled with sterilized field soil, while roots dipped in sterilized water were used as the control. The experiment considered of ten pots for the inoculation treatment and six pots for the control treatment. All pots were maintained in the greenhouse. After 15 days, it was observed that 80% of the inoculated plants displayed symptoms consistent with the field observations, indicating successful infection. In contrast, plants in the control treatment did not exhibit any symptoms. The same fungal pathogen as F. tricinctum CH-2 was reisolated from the diseased root tissue and confirmed through morphological and molecular assays, thereby satisfying Koch's postulates. This is the first documented report of F. tricinctum causing root rot in C. humilis in China. This disease has the potential to become one of the most significant diseases affecting C. humilis in China.

First report of root rot caused by Fusarium nygamai on foxtail millet (Setaria italica) in China.

Foxtail millet [Setaria italica (L.) P. Beauv.] is one of the most important nutritious food crops in China. In August 2020, plants of the foxtail millet cultivar Xiao Huang Miao were found that were wilted and root rot symptoms of 25-75% incidence in a field production area of about 3000 m2 near Tongliao of Inner Mongolia and Chaoyang cities of Liaonning province. The wilted plants showed yellowing, stunting, and the lower stalk became straw colored, softened, with gray-whitemould on the surface of the stem nodes. The root system was poorly developed, brown and rotted. Symptomatic roots were surface-disinfested with 70% ethanol for 1 min and in 2% sodium hypochlorite (NaOCl) for 3 min, rinsed with sterilized water three times, and placed on potato dextrose agar (PDA) and incubated at 26ºC for 5 days. Ten pure cultures were obtained from single conidia with an inoculation needle under stereomicroscope. The cultures were transferred to carnation leaf agar (CLA) medium and incubated two weeks in the dark at 26ºC for microscopic observation. Macroconidia had one to four septa (three septa dominated), and were slender and straight with curved apical cell and foot-shaped basal cell, 25.5 - 30.5 × 2.5 - 4.5 μm (n=50). Microconidia were non-septate, oval, and were formed in short chains or false heads on monophialides, 2.5 - 15 × 2.75 - 4.0 μm (n=50). Chlamydospores were singly or in chains, circular or subcircular, 5.25 - 11.5 μm in diameter (n=50). Morphologically, the fungus was identified as Fusarium nygamai Burgess & Trimboli (Klaasen and Nelson,1998; Leslie and Summerell, 2006,). To validate this identification, rDNA internal transcribed spacer (ITS), partial translation elongation factor 1 alpha (TEF-á) gene, and RNA polymerase II second largest subunit (rpb2) of the ten isolates were amplified and sequenced (White et al.1990;O'Donnell K. et al. 2015,2010). Identical sequences were obtained and the sequence of the isolate GZGF23 was submitted to GenBank. BLASTn analysis of the ITS (OL964384), TEF-á (OL961517) and RPB2(ON756204) sequence of isolate GZGF23 revealed 99.86% (MH862671, 557/565bp), 100% (MT011009, 713/1770bp) and 100% (MT010976, 1002/3907bp) sequence similarity respectively with F. nygamai (CBS749.97). Pathogenicity studies were conducted on outdoor potted ground and with the foxtail millet cultivar "Xiao Huang miao". Five 12-L pots were filled with sterilized field soil mixed with 300ml conidial suspension at 3 × 105 spores/ml. Another five 12-L pots were filled with sterilized field soil mixed with 300ml sterilized water that served as controls. About twenty seeds per pot were surface disinfected in 2% NaOCl for 3 min, and rinsed with sterilized water. The foxtail millet seeds were sown the same day as soil inoculation and 6 plants were left in each pot when seedling emerged. Five weeks after seedling emergence, all inoculated plants exhibited symptoms similar to the syptoms observed in the field but control plants had no symptoms. The same results were obtained when pathogenicity tests were repeated two times in the same manner. Fusarium nygamai was reisolated from inoculated plants and its morphological and molecular characteristics matched the original isolate, but the fungus was not reisolated from control plants. This is the first report of root rot caused by F. nygamai on foxtail millet in China. The disease might bring a threat to foxtail millet production and effective control measures should be identified to reduce losses.

Invasive plants have greater growth than co‐occurring natives in live soil subjected to a drought‐rewetting treatment

Abstract Several studies have shown that invasive plant species respond more negatively to drought than native plant species, but little remains understood of how and whether drought‐rewetting events may affect growth of invasive and co‐occurring native plant species both directly and indirectly through soil microorganisms. In a fully crossed factorial design, we grew individuals of four congeneric pairs of invasive and native plant species in 2.5 L pots that contained live or sterilized field soil under one of three drought treatments: no‐drought, drought, drought‐rewetting. Results show that drought caused a significantly greater decline in total biomass of invasive plant species than that of native plant species regardless of the presence of live soil microorganisms. However, total biomass of the invasive plant species exhibited a greater recovery from drought following rewetting than did that of the native plant species. Moreover, recovery from drought in invasive plant species tended to be stronger in live soil than in sterilized soil, while for the native plant species, recovery from drought was stronger in sterilized soil than in live soil. Overall, these results suggest that in ecosystems that experience cycles of drought and rewetting, invasive plant species may grow larger than co‐occurring native plant species. Moreover, soil microorganisms may facilitate greater recovery from drought in invasive plant species than in co‐occurring native plant species. Read the free Plain Language Summary for this article on the Journal blog.

First Report of Plasmodiophora brassicae causing clubroot on Thlaspi arvense L. in Sichuan Province of China.

Pennycress (Thlaspi arvense L.), an annual herb of the mustard family Brassicacae, is native to Eurasia and now widely distributed throughout temperate North America. This species is currently being developed as a medicinal herb used to treat nephritis in China and an oilseed crop for biofuel production (Roque et al.2012). In November 2020, stunt and wilt symptoms were observed on above ground parts and swollen club-shaped galls were observed on the roots of T. arvense in most of the Chinese cabbage growing area in Kangding (30°03'"N,102°02'"E), Sichuan Province of China. The average disease incidence of swollen roots on T. arvense was 91.2% ( n=80). To identify the causal agent of this disease, the swollen roots of T. arvense were collected, crushed and observed under microscope (Fei et al.2017). Abundant resting spores were found in the root galls, which were spherical and 2.0 to 3.1 μm in diameter with an average length of 2.7 nm (n=100). The healthy roots and the root galls of T. arvense plants were further evaluated by PCR with P. brassicae-specific primers TC2F/TC2R (Cao et al. 2007). The results showed that a DNA fragment with an estimated size of 520 bp, as expected for that of P. brassicae, was consistently amplified in diseased roots, No PCR amplification occurred in the healthy roots with the TC2F/TC2R primers. Blast analysis of the 520 bp segment (GenBankMZ040496) showed the highest identity with the sequence of small subunit ribosomal RNA gene of P. brassicae (GenBankMH762161, 97.7%, E value=0). These results confirmed that the pathogen in the galled roots of T. arvense was P. brassicae. The pathogenicity of isolated P. brassicae was tested on both T. arvense and Chinese cabbage (B. campestris ssp. pekinensis). Resting spores were isolated from the diseased roots (Castlkbury et al. 1994) of T. arvense and suspended in Hoagland's solution to the final concentration of 1 × 107 spores per milliliter. Fifteen plastic pots (10 cm bottom diameter, 16 cm upper diameter, 13 cm high) were filled with soil (1 kg per pot) that was sterilized twice with high-temperature (121℃), high pressure (19 PSI) for 1.5 hours with a time interval of 2 days between. Inoculated pots received 100 mL spore suspension each. Fifteen control pots with sterilized field soil were treated with 100 mL Hoagland's solution each. Seeds of T. arvense and B. campestris were pre-germinated at 20°C on moist filter paper for 7 days and transplanted into the pots, five seedlings each and five pots per treatment. The pots were maintained in a greenhouse with 16 hours photoperiod at 24°C/16°C day/night temperature. After 7 weeks, plants in each pot were uprooted and the roots cleaned in running water and inspected for clubroot symptoms. Plants of T. arvense and Chinese cabbage in pots inoculated with resting spores showed clubroot symptoms while no disease symptoms were observed on any control plants. The disease incidence rate was 95.4% on T. arvense and 81.2% on B. campestris. Therefore, it was confirmed that P. brassicae could cause clubroot disease on T. arvense. To our knowledge, this is the first published report of clubroot disease on T. arvense in China. This finding is helpful for the management of clubroot on herbs and plants of biological origin in the cruciferous family.

First report of Fusarium oxysporum causing root rot of Gentiana scabra bunge (Adenophora capillaris) in China.

Gentiana scabra bunge (Adenophora capillaris) is a traditional Chinese medicine crop in the northeast China. In July 2019, the root rot symptoms of A. capillaris were observed in its production field (approximately 1.3 hectares) iningyuan Manchu Autonomous County (41º47'28" N, 124º21'35" E), Fushun City in Liaoning province, China. Typical symptom included wilting, darkening, and rotting of the root collar and vascular bundle, leading to plant defoliation and death. Approximately 25% of the plants in the field showed the symptoms, with 2-year-old plants having more severe symptoms. Root tissue samples were collected from the diseased plants, surface-sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 5 min, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). After 3 days of incubation at 25 °C, white Fusarium-like colonies grew out from the symptomatic root tissue pieces. Five single-spore isolates were obtained from 10-day-old cultures using single-spore isolating technique. The fungus produced many macroconidia with the typical macroconidia of Fusarium spp. on carnation leaf agar (CLA) after 18 days of incubation at 26°C. They were falculate, slender and slightly curved, and their cells at both ends were sharp. Macroconidia had 3 to 5 septa, measuring 24.8 to 48.6 × 3.5 to 6.4 μm (n=50). Microconidia had 1 to 2 septa, elliptical, rounded tip, measuring 6.7 to 22.5 × 2.4 to 5.5 μm (n=5). Morphologically, the isolates were identified as Fusarium oxysporum (Leslie and Summerell 2006). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and the translation elongation factor-1a (TEF-1α) of Isolate LD528-1 were amplified with the general primer ITS1/ITS4 and TEF-1α primer EF-1/EF-2 (O'Donnell et al. 1998). The resulting sequences were deposited in GenBank (acc. nos. MW418098 and MW423622). BLASTn analysis of the ITS sequence (KU939043) and TEF sequence (MW423622) revealed 99.06% sequence identity with F. oxysporum (KU939043) and 100% with F. oxysporum (MN892354), respectively. For pathogenicity test, a pot experiment was conducted in a greenhouse with 22 to 28°C and 65 to 90% relative humidity. Roots of A. capillaris were dipped in a spore suspension (1×107 conidia/ml) of Isolate LD528-1 for approximately 5 min, and then planted into the pots filled with sterilized field soil. Root dipped in sterilized water served as the controls. There were five pots for the inoculation treatment and three pots for the control treatment. All treated pots were placed and maintained in the greenhouse. After 15 days, 80% of inoculated plants were infected, with the symptoms similar to those observed in the field. The plants in the control treatment did not develop any symptoms. The same fungus was re-isolated from the diseased root tissue and confirmed by morphological and molecular assays as described above. This is the first report of F. oxysporum causing root rot of gentiana scabra bunge in China. This disease can become one of most important diseases in gentiana scabra bunge in China.

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.

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 Bacterial Wilt Caused by Ralstonia solanacearum on Plectranthus amboinicus in Martinique.

Plectranthus amboinicus, commonly known as Gwo ten in the French West Indies (Martinique), is a semi-succulent perennial plant of the Lamiaceae family. This aromatic plant wich is widespread naturally throughout the tropics is of economic importance because of the therapeutic and nutritional properties attributed to its natural phytochemical compounds wich are highly valued in the pharmaceutical industry. In March 2019, wilted P. amboinicus plants intercropped with tomato plants (cv. Heatmaster) in order to reduce the insect-pest damages on tomato, were observed in a field located at the CIRAD experimental station in Lamentin, Martinique (14.663194 N, -60.999167 W). Average disease incidence of 65.74% was recorded on P. amboinicus, in 3 plots with an area of 22.04 m2. The initial symptoms observed were irregular, black, necrotic lesions on leaves. After 10 days, plants wilted and black stripes were observed on stems. Within 4 weeks, more than 50% of plants were fully wilted. Longitudinal stem sections of the wilted plants showed brown vascular discoloration. The cut stems of the wilted plants released a whitish bacterial ooze in water. In all, 108 stem sections were collected, surface disinfected with 70% ethanol and each was crushed in 2 mL of Tris-buffer, then processed for bacterial isolation by plating on modified Semi-Selective Medium from South Africa SMSA (Engelbrecht 1994). Typical Ralstonia solanacearum colonies grew on SMSA medium for 100 of the 108 samples after incubation for 48h at 28°C and were identified as Ralstonia solanacearum using diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all the strains in R. solanacearum Phylotype IIA. A subset of 11 strains was selected for sequevar identification. All the strains were identified as sequevar I-39 (100% nucleotide identity with strain ANT92 - Genbank accession EF371828), by partial egl sequencing (Fegan and Prior 2005) (GenBank Accession Nos. MT314067 to MT314077). This sequevar has been reported to be widespread in the Caribbean and tropical America on vegetable crops (particularly on tomato), but not on P. amboinicus (Deberdt et al. 2014; Ramsubhag et al. 2012; Wicker et al. 2007). To fulfil Koch's postulates, a reference strain, isolated from diseased P. amboinicus (CFBP 8733, Phylotype IIA/sequevar 39), was inoculated on 30 healthy P. amboinicus plants. A common tomato cultivar grown in Martinique (cv. Heatmaster) was also inoculated on 30 plants with the same bacterial suspension. Three-weeks-old plants of both crops grown in sterilized field soil were inoculated by soil drenching with 20 ml of a calibrated suspension (108 CFU/mL). P. amboinicus and tomato plants drenched with sterile water served as a negative controls. Plants were grown in a fully controlled environment at day/night temperatures of 30-26°C ± 2°C under high relative humidity (80%). The P. amboinicus plants started wilting 9 days after inoculation, and within four weeks 60% of the P. amboinicus plants had wilted. The tomato plants started wilting 5 days after inoculation with 62% of wilted plants within four weeks. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. No symptoms were observed and no R. solanacearum strains were isolated from negative controls plants. To our knowledge, this is the first report of R. solanacearum causing bacterial wilt on Gwo ten (P. amboinicus) in Martinique. The importance of this discovery lies in the reporting of an additional host for R. solanacearum, which can be associated with other crops as tomato crop in order to reduce the abundance of insect-pests. Further studies need to be conducted to assess the precise distribution of bacterial wilt disease on P. amboinicus in Martinique and to develop a plan of action avoiding its association with R. solanacearum host crops as tomato for reducing epidemic risk.

First Report of the Root-Knot Nematode, Meloidogyne floridensis, on Tomato in Georgia, USA.

Meloidogyne floridensis, also known as the peach root-knot nematode (RKN), is a new emerging species found to break crop host-resistance to M. incognita (Stanley et al. 2009). It was first described from Florida (Handoo et al. 2004) parasitizing M. incognita-resistant rootstock cultivars of peach (Prunus persica), and tomato (Solanum lycopersicum) (Church 2005). The nematode has recently been reported in California's almond orchards (Westphal et al. 2019) and peach rootstock (cv. Guardian) in South Carolina (Reighard et al. 2019). In a 2018 survey of vegetable fields sampled randomly in South Georgia, RKN was found with a high density (5,264 second-stage juveniles (J2)/100 cm3 of soil) from a tomato field in Ware County, GA. The soil sample consist of 30 soil cores sampled at 20-cm depth across the field in a zig-zag motion. To perform Koch's postulate, 2,000 eggs from a single egg-mass culture were inoculated into deepots filled with mixture of sand and sterilized field soil (1:1 v/v) and grown with tomato cv. Rutgers for 60 days in the greenhouse. A reproduction factor of 21.1 ± 6.1 was obtained confirming the nematode parasitism on tomato (Fig. 1S). For molecular identification, DNA was extracted by smashing three individual females isolated from the galled roots in 50 µl sterile distilled water, followed by a freeze-thaw (95°C, 1 min). Results of PCR analyzes by species-specific primers (Fjav/Rjav, Finc/Rinc and Far/Rar) did not detect the nematode species (Zijlstra et al. 2000). PCR products were obtained and sequenced from two primer sets consisting of the forward NAD5F2 (5'-TATTTTTTGTTTGAGATATATTAG-3') and the reverse NAD5R1 (5'-CGTGAATCTTGATTTTCCATTTTT-3') for amplification of a fragment of the NADH dehydrogenase subunit 5 (NADH5) gene (Janssen et al. 2016), and the forward TRANH (5'-TGAATTTTTTATTGTGATTAA-3') and the reverse MRH106 (5'-AATTTCTAAAGACTTTTCTTAGT-3') for amplification covering a portion of the cytochrome c oxidase subunit II (COII) and large subunit 16SrDNA (16S) gene (Stanton et al. 1997). DNA sequence of NADH5 gene fragment (accession no. MT795954) was 100% identical (532/532 bp) with a M. floridensis isolate from California and South Carolina (accession no. MH729181 and MN072363), while fragment of the COII and 16S genes (accession no. MT787563) was 99.76% identical (421/422 bp) with an isolate from Florida (accession no. DQ228697). The nematode females were also used for morphometric and perennial pattern analysis. Several micrographs with the inverted microscope (ZEISS Axio Vert.A1, Germany) and camera (ZEISS Axiocam 305 color, Germany) were taken from ten J2s for mean, standard deviation and range of body length: 362.7 ± 11.2 (340.4-379) µm, maximum body width: 15 ± 1.3 (12.4-16.4) µm, stylet length: 12.3 ± 1.3 (9.5-14) µm, hyaline tail terminus: 8.9 ± 1.1 (7.5-10.9) µm and tail length: 35.7 ± 4.4 (28.5-39.5) µm. Morphological measurements and configuration of perineal patterns (Fig. 2S) were comparable to previous reports of M. floridensis isolates from Florida (Handoo et al. 2004; Stanley et al. 2009). To the best of our knowledge, this is the first report of M. floridensis in Georgia as the fourth state in the USA after South Carolina, California and Florida. This nematode has been reported to parasitize several vegetable crops, including cucumber, eggplant, tomato, snap bean and squash. Furthermore, RKN resistant cultivars of tomato (harboring Mi-1 gene), pepper (harboring N gene), corn cv. Mp-710 and tobacco cv. NC 95 have been found susceptible to M. floridensis (Stanley et al. 2009), making it a serious threat.

Contribution of pathogenic fungi to N2O emissions increases temporally in intensively managed strawberry cropping soil.

Intensively managed agriculture land is a significant contributor to nitrous oxide (N2O) emissions, which adds to global warming and the depletion of the ozone layer. Recent studies have suggested that fungal dominant N2O production may be promoted by pathogenic fungi under high nitrogen fertilization and continuous cropping. Here, we measured the contribution of fungal communities to N2O production under intensively managed strawberry fields of three continuous cropping years (1, 5, and 10 years) and compared this adjacent bare soil. Higher N2O emission was observed from the 10-year field, of which fungi and prokaryotes accounted for 79.7% and 21.3%, respectively. Fungal population density in the 10-year field soil (4.25 × 105 colony forming units per g (CFU/g) of air-dried soil) was greater than the other cropping years. Illumina MiSeq sequencing of the nirK gene showed that long-term continuous cropping decreased the diversity of the fungal denitrifier community, but increased the abundance of Fusarium oxysporum. Additionally, F. oxysporum produced large amounts of N2O in culture and in sterile 10-year field soil. A systemic infection displayed by bioassay strawberry plants after inoculation demonstrated that F. oxysporum was a pathogenic fungus. Together, results suggest that long-term intensively managed monocropping significantly influenced the denitrifying fungal community and increased their biomass, which increased fungal contribution to N2O emissions and specifically by pathogenic fungi. KEY POINTS: • Distinguishing the role of fungi in long-term continuous cropping field. • Identifying the abundant fungal species with denitrifying ability.

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

Maize [Zea mays L.] is animportantfood 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.

Abiotic and Biotic Soil Legacy Effects of Plant Diversity on Plant Performance

Soil legacies are typically examined for individual plant species, and we poorly understand how soil legacy effects created by entire plant communities influence plant growth. We used soils collected from a biodiversity field experiment to examine how the soil legacy effects of plant diversity influence the growth of a focal plant species. In the field, we experimentally assembled and maintained grassland communities (0, 1, 2, 4 or 9 species) for two years. We collected soil from all plots and examined the growth of Jacobaea vulgaris in these soils under controlled conditions, and compared this to the performance of individuals that were planted directly in the plots. J. vulgaris was not part of the species pool used in the biodiversity experiment, but commonly occurs in the area. To disentangle different components of the legacy effects (soil nutrients vs soil biota), in the pot experiment we tested the effects of plant growth in pure field soil and in sterilized background soil inoculated with live or with sterilized field soil. We found a weak positive legacy effect of plant diversity on J. vulgaris root biomass, but only in pure field soil and not in the inoculated treatments. Interestingly, for individuals planted in the field plots, plant biomass was negatively related to the diversity of the surrounding plant community but this was mainly due to high biomass in bare plots. In the pot experiment, plant biomass also varied among soils collected from different monocultures. Soil fungal community composition was not affected by the diversity of the plant community, but the biomass of the plants grown in pots with pure field soil correlated with fungal composition. The biomass of plants grown in pure field soil was also positively correlated with nitrogen availability in the soil, and negatively with the cover of three plants species in the communities. In conclusion, our study does not provide strong evidence for an important role of plant diversity on soil legacy effects on J. vulgaris, and shows that for this plant species, performance is related to both the biotic and abiotic characteristics of the soil in which it grows.

Interactions between putatively endophytic bacteria and tall fescue (Festuca arundinacea): plant growth promotion and colonization in host and non-host cultivars

Tall fescue (Festuca arundinacea) is a forage grass highly used in prairies. Cultivar SFRO Don Tomas has excellent drought and cold resistance, characteristics which are influenced by the native endophytic bacterial community. The aims of this work were to deepen the characterization of a selected group of putatively endophytic strains with different plant growth promoting (PGP) traits and to analyze the relationship between colonization and PGP abilities. PGP with single inoculations in pots containing a mixture of sterile sand and field soil and with paired inoculations in field soil cores were carried out. Colonization of plants inoculated singly or co-inoculated under gnotobiotic conditions was quantified. Confocal laser microscopy was used to visualize the colonization and infection processes of strain Streptomyces sp. UYFA156 in cultivars SFRO Don Tomas and Tacuabe. Tall fescue cultivars displayed different responses to single bacterial inoculation. Inoculation with two strain pairs which included UYFA156 were successful in promoting growth in soil cores. Higher bacterial densities did not correspond to higher PGP. Strain UYFA156 was observed colonizing both fescue cultivars. UYFA156 is a true endophyte of tall fescue. Its PGP activity is cultivar-specific and may be related to the modulation of other endophytes.

First Report of Bacterial Wilt of Amaranth (Amaranthus cruentus) Caused by Ralstonia solanacearum in Benin

Amaranth (Amaranthus cruentus) is an important leafy vegetable cultivated in Benin. In September 2017, wilted amaranth plants of accessions AC NL and Madiira-1 that did not exhibit any foliar discoloration were observed at a field in Cotonou, Benin. Average disease incidences of 72.4 and 27.08% were recorded on accessions AC NL and Madiira-1, respectively, in two plots with an area of ∼8.5 m². The longitudinal sections of most stems of the infected plants showed vascular discoloration, and all the cut stems released whitish bacterial ooze in water. On semiselective modified medium from South Africa (SMSA) agar plates (Engelbrecht 1994), isolated bacterial colonies were morphologically similar to Ralstonia solanacearum. Koch’s postulates were performed with three bacterial isolates by inoculating the susceptible amaranth accession AC NL grown in sterilized field soil. Amaranth plants (10 per isolate) were inoculated by drenching the soil around the crown region of the plant with 40 ml of bacterial suspension (10⁸ CFU/ml). Susceptible tomato cultivar (cv. Tohounvi; five per isolate) was also inoculated with the same bacterial suspensions. Amaranth and tomato plants drenched with sterile distilled water served as the negative control. Three-week-old plants were inoculated and maintained under shade at about 28 to 31°C. Amaranth plants started wilting 12 days after inoculation, and within 4 weeks 73.3% of plants wilted, and all the inoculated tomato plants wilted within 7 days. The noninoculated amaranth and tomato plants remained healthy during the period of the experiment. R. solanacearum was recovered from all symptomatic plants on SMSA medium. Diagnostic polymerase chain reaction (PCR) was conducted using R. solanacearum species-specific primer pairs 759/760 (Opina et al. 1997). The size of the PCR product (282 bp) of the samples from amaranth suggested that the pathogen was R. solanacearum. Multiplex PCR using specific primer for phylotypes I, II, III, and IV (Fegan and Prior 2005) identified the amaranth strain as R. solanacearum phylotype I. Endoglucanase partial gene amplification and sequencing were conducted using the Endo primer pairs (Gutarra et al. 2017). The BLAST search of DNA sequence of the amplified Endo gene region confirmed that the strain was 99% identical to NCBI accessions AB508612 and HM775371 and 98% identical to MF783890. The nucleotide sequence was submitted to the NCBI GenBank and available with the accession number MH397250. Surveys to evaluate the impact of bacterial wilt disease to amaranth farmers in Benin and a plan of action for crop rotation for reducing risk are planned. This is the first recorded report of R. solanacearum causing bacterial wilt on amaranth in the Republic of Benin.

Do soil biota influence the outcome of novel interactions between plant competitors?

1. Species are shifting their ranges, for example to higher elevations, in response to climate change. Different plant species and soil microbiota will likely shift their ranges at different rates, giving rise to novel communities of plants and soil organisms. However, the ecological consequences of such novel plant-soil interactions are poorly understood. We experimentally simulated scenarios for novel interactions arising between high- and low elevation plants and soil biota following asynchronous climate change range shifts, asking to what extent the ability of plants to coexist depends on the origin of the soil biota. 2. In a greenhouse experiment, we grew pairs of low- (Poa trivialis and Plantago lanceolata) and high- (Poa alpina and Plantago alpina) elevation plant species alone and against a density gradient of con- or heterospecific neighbours. Plants grew on sterilized field soil that was inoculated with a soil community sampled from either low- or high elevation in the western Swiss Alps. We used the experiment to parameterize competition models, from which we predicted the population-level outcomes of competition in the presence of the different soil biota. 3. In the absence of neighbours, three of the four species produced more biomass with the low elevation soil biota. As a result of generally similar responses across plant species, soil biota tended not to affect plant interaction outcomes, with the low elevation species generally predicted to competitively exclude high elevation species irrespective of the soil biota origin. However, the low elevation grass Poa trivialis was only able to invade communities of Poa alpina in the presence of a low elevation soil biota. This suggests that, at least in some cases, the outcome of novel competitive interactions between plants following climate change will depend on whether shifts in the distribution of plant and soil organisms are asynchronous. 4. Synthesis. Our results indicate that the changing soil communities that plants encounter during range expansion can influence plant performance. However, this is only likely to alter expectations for the ability of plants to coexist following climate change if plant species respond differently to the change in the soil community.

Soil pathogen-aphid interactions under differences in soil organic matter and mineral fertilizer.

There is increasing evidence showing that microbes can influence plant-insect interactions. In addition, various studies have shown that aboveground pathogens can alter the interactions between plants and insects. However, little is known about the role of soil-borne pathogens in plant-insect interactions. It is also not known how environmental conditions, that steer the performance of soil-borne pathogens, might influence these microbe-plant-insect interactions. Here, we studied effects of the soil-borne pathogen Rhizoctonia solani on aphids (Sitobion avenae) using wheat (Triticum aestivum) as a host.In a greenhouse experiment, we tested how different levels of soil organic matter (SOM) and fertilizer addition influence the interactions between plants and aphids. To examine the influence of the existing soil microbiome on the pathogen effects, we used both unsterilized field soil and sterilized field soil.In unsterilized soil with low SOM content, R. solani addition had a negative effect on aphid biomass, whereas it enhanced aphid biomass in soil with high SOM content. In sterilized soil, however, aphid biomass was enhanced by R. solani addition and by high SOM content. Plant biomass was enhanced by fertilizer addition, but only when SOM content was low, or in the absence of R. solani.We conclude that belowground pathogens influence aphid performance and that the effect of soil pathogens on aphids can be more positive in the absence of a soil microbiome. This implies that experiments studying the effect of pathogens under sterile conditions might not represent realistic interactions. Moreover, pathogen-plant-aphid interactions can be more positive for aphids under high SOM conditions. We recommend that soil conditions should be taken into account in the study of microbe-plant-insect interactions.

Propagation technique of arbuscular mycorrhizal fungi isolated from coastal reclamation land

Arbuscular mycorrhizal fungi (AMF) are well known for their plant growth promoting potential and stress tolerance ability. AMF promotes plant growth even at adverse environmental conditions and improve plant nutrient uptake. This study aimed to propagate AMF using a single spore inoculation technique. Soil samples were collected from salt affected Saemangeum reclaimed land. Collected soil samples were subjected to soil analysis. In the single spore inoculation method, sorghum sudangrass (Sorghum bicolor L.) was inoculated with a single spore. Among the 150 inoculants, six spores were able to germinate in vitro. Geminated spores were transferred to 1 kg pots containing sterilized field soil. After 120 days, the contents were mixed and transferred to 2.5 kg pots and maintained for another 120 days. After 240 days, spore count and colonization were checked. The propagated spores were identified using nested PCR followed by sequencing. The 18S rDNA sequencing of spores revealed that the spores belonged to Gigaspora margarita and Claroideoglomus lamellosum. Among the 6 inoculants, Claroideoglomus lamellosum S-11 (391 spore per 100 g of soil) had the highest spore count followed by Gigaspora margarita S-23 (235 spore per 100 g of soil). Slide method allowed visual monitoring of spore germination in vitro as well as being able to mass produce pure cultures of AMF for bio-inoculation purposes.

Field method to monitor the mycoparasitic fungus Coniothyrium minitans

We designed specific primers for Coniothyrium minitans, a sclerotia-parasitizing biocontrol agent (BCA), and quantified its biomass in the field by quantitative polymerase chain reaction. Because chemical properties of the soil can decrease the DNA extraction efficiency and DNA polymerase activity, standard curves were calibrated by adding known concentrations of BCA to sterilized field soil. The BCA biomass was monitored over time and after various treatments. Because the distribution of the BCA biomass was uneven in the field, a method to spread BCA uniformly in the field is needed.