Root Discoloration Research Articles

Assessing Pathogenicity and Cultural Characteristics of Fusarium oxysporum f. sp. ricini Isolates from Major Castor Growing Regions of India

The present study was undertaken to investigate the pathogenicity and cultural characteristics of 20 isolates of Fusarium oxysporum f. sp. ricini, the causal fungus of wilt disease of castor (Ricinus communis L). Pathogenicity tests on the susceptible cultivar JI-35 revealed that all isolates were pathogenic, with symptoms progressing from yellowing and drooping of leaves to marginal necrosis, wilting and root discoloration. The incubation period varied significantly among the isolates, ranging from 10.0 to 22.5 days, while the per cent disease incidence (PDI) ranged from 51.7% to 100%. Cultural characterization that includes mycelial colour, colony morphology, pigmentation, growth habit and sporulation highlighted significant variability among the isolates of F. oxysporum f. sp. ricini. The colour of mycelium among the isolates ranged from white to various shades of pink and yellow, with growth habits observed as fast, moderate, or slow. Sporulation rates varied from very high to sparse among the isolates. The observed diversity in pathogenicity and cultural traits emphasized the presence of genetic variability in F. oxysporum f. sp. ricini isolates. These results are vital for understanding the pathogen virulence and for developing effective management strategies for wilt disease in castor crop.

First Report of Wilt Caused by Fusarium nanum (FIESC 25) on Mungbean (Vigna radiata) in Pakistan.

Mungbean, Vigna radia (L.) R. Wilczek, is ranked 2nd next to chickpea (Cicer arietinum) in total cultivation and production in Pakistan. In August of 2022 and 2023, mungbean plants (cv. PRI Mung-2018) were found wilting in a field at the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Wilted leaves turned yellow, died, but remained attached to the stem. Vascular tissue at the base of the stem showed light to dark brown discoloration. Roots were stunted with purplish brown to black discoloration. Symptomatic mungbean plants were collected from fields at five different locations (20 samples/location). Disease incidence was similar among the five fields, ranging from 5 to 10% at each location depending upon type of germplasm and date of sowing. For fungal isolation and morphological identification, symptomatic stem and root tissues were cut into ~5 mm2 pieces with a sterilized blade. Tissues were surface-sterilized for one min in a 0.5% sodium hypochlorite solution, rinsed twice in sterilized water, air dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) containing 0.5 g/L-1 streptomycin sulphate. Plates were incubated for 3-4 days at 25 ± 2°C with a 12-h photoperiod. Single-spore cultures were used for morphological and molecular analyses. Isolates on PDA grew rapidly and produced abundant white aerial mycelium that turned off-white to beige with age. Macroconidia were hyaline, falcate, typically 3-to-6 septate with a pointed apical cell and a foot-shaped basal cell, measuring 24.5-49.5 x 2.7-4.7 μm (n = 40). Globose to obovate chlamydospores measuring 5.8 ± 0.5 μm (n = 40) were produced singly or in chains and were intercalary or terminal and possessed roughened walls. The morphological data indicated the isolates were members of the genus Fusarium (Leslie and Summerell 2006). To obtain a species-level identification, a portion of translation elongation factor 1-α (TEF1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2) region were PCR amplified and sequenced using EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (Hofstetter et al. 2007), and 5f2/7cr (Liu et al. 1999) primers, respectively. DNA sequences of these genes were deposited in GenBank under accession numbers MW059021, MW059017 and MW059019, respectively. The partial TEF1, RPB1 and RPB2 sequences were queried against the Fusarium MLST database (https://fusarium.mycobank.org/page/Fusarium_identification), using the polyphasic identification tool. The BLASTn search revealed 99.9% identity of the isolate to F. nanum (Xia et al. 2019), formerly FIESC 25 of the F. incarnatum-equiseti species complex (MRC 2610, NRRL 54143; O'Donnell et al. 2018). To confirm pathogenicity, roots of 3-5 leaf stage mungbean seedlings were soaked in a 106 spores ml-1 conidial suspension of the fungus for 15 min and then planted in 10 cm pots containing sterilized soil. Mock-inoculated plants with sterile water served as a negative control. Twenty pots that were used for each inoculated and control treatment were maintained at 25 ± 2°C, 14:8 h photoperiod, and 80% relative humidity in a growth chamber. After 15 days, leaf yellowing, internal browning from the base of stems and root discoloration was observed in all the inoculated plants. The uninoculated negative control plants remained asymptomatic. Fusarium nanum was re-isolated from artificially inoculated plants and identified by colony growth, conidial characteristics on PDA and molecular analyses (TEF1). To our knowledge, this is the first report of wilt caused by F.nanum on mungbean in Pakistan. In Pakistan, mungbean cultivation in irrigated areas has increased in recent years. It has been introduced frequently in citrus orchards, crop rotation of maize and sesame, intercropping with sugarcane and as green manure. However, citrus, maize, sesame and sugarcane are also hosts of Fusarium spp. Therefore, this information warrants sustainable crop protection and may have an impact on further interaction of F. nanum with other wilt pathogens.

Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea

BackgroundAphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches.ResultsIn this study, we inoculated roots of the pea (P. sativum L.) genotypes ‘Linnea’ (susceptible) and ‘PI180693’ (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype ‘PI180693’, Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor.ConclusionsThis study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation.

First report of Root rot in Cerasus subhirtella Caused by Fusarium solani and Fusarium oxysporum in China.

Cerasus subhirtella (Miq.) Sok. is a widely used ornamental tree in urban areas around China and has a high ornamental value. From 2018 to 2020, a root rot disease was observed in C. subhirtella in Meitan County, Weng'an County, and Guiyang city of Guizhou, China (106.71 E, 26.57 N). Diseased C. subhirtella trees exhibited wilting with leaf chlorosis accompanied by brown to black root discoloration. In an area of 100 ha in total, with disease incidence ranging from 60 to 80%. Six symptomatic plants with root rot were randomly collected from three locations where disease symptoms were observed for pathogen isolation. Fifty fragments of diseased roots (5×5mm) were disinfected in 3% sodium hypochlorite for 30 s and 75% alcohol for 60 s, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA; BoWei, Shanghai), and incubated at 28 °C in the dark for 7 days. Eighteen isolates were purified by single spore culturing. Typical Fusarium spp. colonies were obtained from all root samples. On PDA, the colonies showed white and the hyphae were dense, while the colony of isolate YH15 showed pale yellow on the back, radial growth and produced chlamydospores. The macrospores (YH15) were straight to subarcuate, measured 15.3 to 25.1 × 2.5 to 6.2 μm (n=50). The microconidia (YH26) were ellipsoid to ovoid, measured 8.6 to 12.7 × 1.6 to 5.1 μm (n=50). These morphological characteristics were consistent with Fusarium spp., as described recently in Vitullo et al. (2014). To confirm the morphological diagnosis, genomic DNA from the isolates was extracted. The internal transcribed spacer (ITS) (White et al, 1990) region of rDNA and a β-tubulin (Varga et al, 2011) gene fragment were amplified with the primers ITS1/ITS4 and Bt2a/Bt2b, respectively, and were subsequently sequenced. Maximum likelihood analysis was carried out using MEGA 11.0. BLAST analysis revealed that the ITS and β-tubulin sequences of isolate YH15 were 100% homologous with F. oxysporum, and the isolate YH26 had a 99.69~100% homology with F. solani. Sequences of isolate YH15 and YH26 were deposited in GenBank (ITS: OQ363005 and OQ363049; β-tubulin: OQ398187 and OQ398180). The isolate YH15 was thus identified as F. oxysporum by the morphological characteristics and sequences analysis, and the isolate YH26 was identified as F. solani. A reconstructed phylogenetic tree also confirmed their phylogenetic position. The healthy 2-year-old C. subhirtella plants grown in autoclaved acid yellow soil were used for the pathogenicity tests. Then, 50 mL of conidial suspension (2.0×105 conidia/mL, in medium) of 7-day-old isolates YH15 and YH26 were gently applied to the soil in each of the 10 pots as the treatment. A sterilized fungal culture matrix (PDB; BoWei, Shanghai) was applied to each of 10 pots as a control. All pots (30 cm high, 25 cm upper diameter, 15 cm base diameter) were placed in a greenhouse (25 °C, 12 h photoperiod). After 30 days of inoculation, all plants inoculated with the isolates showed wilting symptoms, and the roots showed light-brown to dark-brown lesions. No symptoms were observed in the controls. The pathogen was reisolated from all symptomatic roots and identified as F. oxysporum and F. solani as described above. The pathogenicity test was repeated twice with similar results. Although this fungus was previously reported to cause root disease in many hosts (Li et al., 2020; Gibert et al., 2022), this is the first report of F. oxysporum and F. solani causing root rot in C. subhirtella in China.

Characterization and pathogenicity of Fusarium species causing sugar beet root rot in Morocco

AbstractThe sugar beet crop ranks second globally among the plant species grown mainly for sugar production. In Morocco, the area under sugar beet farming occupies approximately 57,000 ha yielding more than 3 million tons of roots. However, sugar beet root‐tip rot (RTR) caused by Fusarium spp. dramatically reduces the anticipated yields, the purity of the resulting juice, and the sugar concentration. The current study aimed at identifying and characterizing the Fusarium species responsible for the root rot in sugar beet grown in the Khenifra‐Beni Mellal region of Morocco. In this survey, 69 isolates of Fusarium were sampled from sugar beet roots showing typical symptoms of root rot from 2019 to 2021. After screening based on the pathogenicity test, 28 isolates were selected and identified based on morphological features and sequence analyses of the ribosomal internal transcribed spacer (ITS) region and translation elongation factor 1 α 34 (TEF‐1 α). Fusarium oxysporum was the most frequently identified species, followed closely by F. solani, F. equiseti, F. nygmai, F. brachygibbosum, F. proliferatum, F. culmorum, and F. falciforme. Six weeks after inoculations under greenhouse conditions, the studied isolates caused internal vascular discoloration and tip rot of sugar beet roots, with disease incidences ranging from 37.5% to 100.0% and a disease index between 30.3% and 70.5%. Isolates belonging to F. solani were the most aggressive. Moreover, the majority of isolates significantly reduced plant growth. To our knowledge, this research article is the first report of Fusarium species inducing RTR in sugar beet in Morocco.

First Report of Fusarium acuminatum Causing Dianthus chinensis root rot and foliage blight in China.

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. In May 2020, a disease was found at several landscape sites in Xuanwu District, Nanjing, China, causing symptoms of foliage blight and root discoloration onapproximately 52% of one-year old D. chinensis plants. To recover the causal pathogen, samples of infected roots and leaves were cut into 5×5 mm2 pieces, surface-disinfected in 75% ethanol for 30 sec, followed by 1% NaClO for 90 sec, rinsed with sterile water three times and placed on potato dextrose agar (PDA) with 0.1 mg/mL of ampicillin at 25 ⁰C. Hyphae growing on PDA were visible from both root and leaf tissues after three days. Individual hyphal tips were transferred to new PDA plates to obtain pure isolates. Three representative isolates were deposited in the China Forestry Culture Collection Center (CFCC 57545,57546, 57547). The hyphae grew radially, densely, and the aerial hyphae were velvety, white, yellow-white, or pink-white. Representative isolate Facu-DCY5 produced three types of conidia (microconidia, macroconidia, and chlamydospores). Macroconidia were sickle-shaped, measuring 25.7-55.4 µm × 3.2-4.6 µm (n=50). Microconidia were numerous, oval or kidney-shaped, measuring 6.8-11.9 µm × 3.5-4.8 µm (n=50). Conidia produced in the aerial mycelium were 16-34 × 2.2-5.3 µm (n=50). The ITS region, TEF1, calmodulin (CMDA), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4, EF1/EF2, CL1/CL2A and 5F2/7CR , respectively and sequenced at Sangon Biotech (Nanjing, China). The ITS sequence of isolate Facu-DCY5 (GenBank No. ON307073.1) was identical to HQ165938.1, ON306850.1, OM964482.1. TEF1 (ON331997.1) was identical to LC546967.1, HQ165866.1, MZ158155.1. CMDA (ON331996.1) was identical to HQ412345.1, MZ921595.1 and MZ921597.1. RPB2 (ON331995.1) was identical to MZ997370.1. Maximum parsimony and maximum likelihood phylogenies of the Facu-DCY5 multilocus sequence data and those of several species within the F. tricinctum species complex identified the isolate from D. chinensis as F. acuminatum . Pathogenicity tests were performed using a conidial suspension (104conidia/mL). Each plant (approx. 0.3 m in -height) was inoculated with 1 mL of the conidial suspension by mixing it into the potting soil (500 g). Control plants were treated with sterile distilled water. All inoculated plants (n=9) in three repeats of the assay exhibited foliage blight and root rot after 15 days, whereas all control plants (n=9) remained asymptomatic. Fusarium isolates with identical morphological features and molecular marker sequences to those of Facu-DCY5 were recovered from foliage blight and root tissues of all the inoculated plants. In China, F. acuminatum has been reported as a pathogen of Cucurbita maxima, Actinidia arguta, Polygonatum odoratumand Schisandra chinensis. This is the first report of F. acuminatum on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nurseries and landscaping industries, we recommend that diseased plants be removed to prevent the spread of F. acuminatum, and that identification of the infecting isolates from D. chinensis at other sites and landscape locations be performed.

Hybrid Genome Assembly of Berkeleyomyces rouxiae, an Emerging Cannabis Fungal Pathogen Causing Black Root Rot in an Aeroponic Facility.

The resurged interest in cultivation of Cannabis sativa has presented an array of new challenges. Among them are the difficult-to-control pests and pathogens that infect cannabis plants. The limited methods for disease control available to cannabis growers necessitates early detection of plant pathogens, something that molecular techniques such as DNA sequencing has greatly improved. This study reports for the first time the fungal plant pathogen Berkeleyomyces rouxiae causing black root rot in high THC-containing cannabis. Aeroponically grown cannabis plants at a licenced production facility in Cranbrook BC, Canada, rapidly displayed root discoloration and rot symptoms despite testing negative for all commercially available pathogen tests. Developing sequencing-based disease diagnostics requires genomic information, so this study presents the first whole genome sequence of the multihost, widespread black root rot pathogen B. rouxiae. Hybrid genome assembly using Oxford Nanopore long-reads and Illumina short-reads yielded a genome size of 28.2 Mb represented over 404 contigs with an N50 of 267 kb. Genome annotation predicted 6,960 protein-coding genes with 59,477 functional annotations. The availability of this genome will assist in sequence-based diagnostic development, comparative genomics, and taxonomic resolution of this globally important plant pathogen.

Identification and Pathogenicity of the Soybean Root Rot Pathogen in Arid Conditions

Soy is an important oilseed crop in Kazakhstan. However, its yield is significantly reduced due to an increase in the development of fungal diseases with soil infection. This study aims to identify the root rot pathogen in soybean varieties in Western Kazakhstan and assess its pathogenicity. Samples of soybean roots to determine the root rot pathogen were selected at the experimental plots of the Aktobe agricultural experimental station in 2021. Genetic analysis of isolates observed on potato dextrose agar medium from soybean root slices was carried out using the sequencing reaction of internal transcribed spacer region nucleotide sequences using the Big Dye Terminator protocol (applied biosystems) with a further comparison of data with the Gen bank database. A test for the pathogenicity of the identified isolates of the fungus was carried out. Statistical data processing was carried out using the R-studio software according to the nonparametric Kruskal Wallis test. Phylogenetic analysis of samples of soybean affected by root rot showed that the main biotic agent causing root rot was the imperfect fungus Fusarium equiseti. Sequencing of the highly informative internal transcribed spacer region of fungal isolates allowed identifying strains 1 and 2 belonging to the species Fusarium equiseti. The pathogenicity test established that isolates of the Fusarium equiseti fungus caused discoloration of seedlings and plant roots and necrosis in soybean seedlings.

First report of Fusarium equiseti causing leaf spots of Bitter gourd (Momordica charantia) in Pakistan.

Bitter gourd (Momordica charantia L.) is an important vegetable crop of the Cucurbitaceae family widely cultivated in Pakistan and around the world. In October 2020, a nutrition management trial of Bitter gourd cv. Seminis-200) was conducted on an area of 10,860 sq. ft. (99×110 feet) at the Agricultural Research farm of Bahauddin Zakariya University, Multan (30.2601° N, 71.5158° E), Pakistan. Symptoms of large, brown necrotic leaf spots were observed on the leaves of bitter gourd vines. The disease started from the yellowing of leaves within the reticulate venation and turned brown. Irregular brown leaf spots coalesced to form large necrotic areas followed by foliar chlorosis then wilting that occurred very late. There were no crown rot symptoms although there was slight discoloration of roots and when cut longitudinally, browning of tissues was observed. The disease was assessed visually with 37% incidence which resulted in poor quality and yield in terms of reduced size and yellowing of fruit. Infected vines along with the roots were collected for the isolation of pathogen. A total of 34 leaves and 22 root samples were collected from the field for isolation. The leaf, collar and root portions were cut into 0.5 to 1 cm in length and surface disinfected with 1% sodium hypochlorite (NaOCl) for 2-3 minutes followed by washing twice with autoclaved distilled water and after drying, placed on potato dextrose agar (PDA) medium, and incubated at 25±2 °C for one week. The fungal colonies of fluffy white growth with light orange pigment were isolated. For morphological characterization, a total of 4 pure cultures were isolated from leaves, collar region and root by single spore technique on carnation leaf agar (CLA) medium after 15 days of incubation at 25±2℃. Curved and thick-walled macroconidia with elongated or pointed apical characteristic foot-shaped basal cells were produced in sporodochia. Macroconidia with 5-7 septa measured 22.50-41.80 μm × 2.90-4.20 μm (n = 60). Thick, brown with roughened walls and subglobose ellipsoidal chlamydospores were observed in clumps or chains with the dimension of 5.8 to 10.8 μm (n = 20). On morphological characteristics, the fungus was identified as Fusarium equiseti (Corda) Sacc. according to Leslie and Summerell (2006). Two single spore isolates were used for molecular identification by amplifying ribosomal DNA of the internal transcribed spacer (ITS) region with ITS1/ITS4 primers (White et al. 1990) and for β-tubulin gene region, primers T1/Bt-2b (O'Donnell and Cigelnik, 1997) were used. The obtained sequences were deposited in GenBank with accession numbers MW880179 and MW880198 from the ITS region and BLAST search in GenBank showed 100 and 98.11% alignment with previously published sequences of F. equiseti with accessions OM992323.1and MT558569.1 respectively. Accession number OM867571from the β-tubulin region showed 100% sequence similarity with F. equiseti with accession MN653163.1. For pathogenicity, macroconidia from 2-week-old cultures on CLA medium were harvested to prepare spore suspension (1 × 106 conidia/ml). Koch's postulates were confirmed on nine bitter gourd plants (cv. Seminis-200) by applying spore suspension of fungal inoculum at 3-4 leaf stage separately on leaves by automizer, on collar region after making incision spore suspension was applied and in the root zone, 20ml spore suspension was added whereas distilled water was used as a control with three replications. Plants were kept under controlled conditions in the greenhouse with 65% to 75% humidity and the temperature was maintained at 32±2 °C for one week. After 7-8 days, inoculated plants began to exhibit symptoms of brown, necrotic leaf spots on the leaves of bitter gourd vines followed by yellowing of leaves that eventually turned brown. Roots showed slight discoloration and browning of vascular bundles and finally, the plants wilted after four weeks. while control plants remained symptomless. The symptoms resembled those noticed in the field. The fungus was re-isolated from leaves, collar region and roots, followed by morphological identification, and finally confirmed as F. equiseti. To the best of our knowledge, this is the first report of a leaf spot caused by F. equiseti in a bitter gourd from Pakistan. If the disease is not managed properly, it may cause a drastic effect on yield under favorable environmental conditions. The pathogen may also damage other cucurbitaceous crops cultivated in the area.

First Report of Crown and Root Rot Caused by Fusarium oxysporum on Photinia × fraseri Dress in China.

Photinia × fraseri Dress was introduced to China in 1998 and has been widely used in gardens as an ornamental plant. From April 2021 to 2022, a diseasecausing blight and root discoloration in approximately 80% of P. × fraseri at several landscape sites in Xuanwu Lake Park. Symptomatic root tissues were immersed in 75% ethanol for 30 s followed by 1% NaClO for 90 s, rinsed with sterile water 3 times, and placed on Potato Dextrose Agar (PDA). After 3 days of dark incubation at 25 °C, white Fusarium-like colonies grew out from the symptomatic root tissue pieces. Three representative isolates (SG1, SG6, and SG23) were obtained and deposited in China's Forestry Culture Collection Center. The hyphae grew radially and the aerial hyphae were velvety, white or pinkish-white . After 20 days, macroconidia, microconidia, and chlamydospores were produced in the colonies on PDA. Macroconidia are sickle-shaped, slightly curved, 23-50.6 µm × 4-6 µm in size. Microconidia were numerous, oval or kidney-shaped, 6.7-12.6 µm × 3.5-5.7 µm in size, with germinating from one or both ends (Fig S1E, F). Chlamydospores were spherical, smooth, and round, in chains or solitary in hyphae (Fig S1G). All three isolates had identical morphological features. Phylogenetic analysis of concatenated CAMD, RPB2 and TEF1 sequences showed that the three isolates clustered in the same clade as F. oxysporum. Two-year old P. × fraseri potted seedlings (30-cm tall, n=12) were placed at the greenhouse (temperature; 26°C, daylight; 14 hours) for the pathogenicity tests . Roots of P. × fraseri were dipped in a 10 mL of the conidial suspension (106 conidia/mL) of each isolate for 2 hours, and the control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated seedlings (n=9) showed crown and root rot . In contrast, none of the control seedlings (n=3) were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Globally, this is the first report of F. oxysporum causing crown blight and root rot of P. × fraseri, which is also a potential threat to the two parent hosts (P. serratifolia and P. glabra). Additional surveys are being conducted for mapping the distribution of F. oxysporum in the Nanjing Province of China.

Effects of harvest period, storage, and genotype on postharvest physiological deterioration responses in cassava

Abstract. Rahmawati RK, Khumaida N, Ardie SW, Sukma D, Sudarso. 2021. Effects of harvest period, storage, and genotype on postharvest physiological deterioration responses in cassava. Biodiversitas 23: 100-109. Postharvest physiological deterioration (PPD) is the major constraint in cassava root production. The breeding program to develop PPD tolerant cassava varieties requires a long period to complete. Although it is the first step in breeding for PPD tolerance, evaluating cassava germplasm responses for the PPD remains a major problem because it is a laborious process, and the evaluation often contains a high experimental error. This study aims to develop methods for evaluating PPD response, i.e., evaluating the effects of two harvest periods and storage on PPD responses of nine cassava genotypes. The developed scoring system based on cassava root discoloration could group the evaluated cassava genotypes into either PPD tolerant, medium tolerant, or sensitive. The tolerant varieties showed less than 10% root discoloration areas, while the medium tolerant was between 11-20%, and the sensitive was larger than 20%, respectively. ADR-24 and GJ-11 were identified as PPD tolerant, while ML-19 was sensitive using the developed scoring system. This study showed that PPD is a complex phenomenon associated with genetics and environmental factors. Root dry matter content and maximum root diameter traits may play an important role in PPD development in cassava. We have developed procedures for identifying genotype responses to PPD and showed that cassava roots harvested as early as eight months after planting and stored as late as five days under the control relative humidity were good conditions for studying PPD. Evaluating the percentages of root discoloration was a good measure for PPD response prediction. Moreover, we showed that less than 10%, 20%, and more than 20% of root discoloration might be used to group cassava into PPD tolerant, medium tolerant, and sensitive for Indonesian accessions. Therefore, the methods developed in this study may support the Indonesian cassava breeding program and provide biochemical and molecular analysis materials to elaborate the mechanisms of PPD in cassava roots.

Characterization of Aphanomyces euteiches Pathotypes Infecting Peas in Western Canada.

Aphanomyces root rot, caused by the soilborne oomycete Aphanomyces euteiches Drechs., has developed into a serious disease in the pea- and lentil-producing areas of the Great Plains of North America. Based on six pea differentials previously used to differentiate 11 pathotypes in France, pathotypes were identified among field isolates from Saskatchewan (14) and Alberta (18). Four isolates from the U.S.A. and standard isolates for pathotypes I and III designated in the French study were also included. Each isolate was tested twice in replicated experiments by inoculating French pea differentials 'Baccara', 'Capella', MN 313, 902131, 552, and PI 80693, along with the Canadian susceptible pea cultivar 'CDC Meadow' and partially resistant USDA line PI 660736 under controlled conditions. Pea plants grown in vermiculite were inoculated 10 days after seeding by pipetting 5 ml of a suspension containing 1 × 103 zoospores ml-1 to the base of each plant. Root discoloration was scored 10 days postinoculation using a 0 to 5 scale. Testing revealed that 38 of the isolates, including standard pathotype I isolate RB84, belonged to pathotype I; four isolates including standard pathotype III isolate Ae109 were pathotype III; and U.S.A. isolate Ae16-01 was a pathotype II isolate. An alfalfa isolate from Quebec was avirulent on all pea genotypes. These findings indicate that pathotype I is predominant on the Canadian prairies.

First Report of Root Rot of Tobacco Caused by Fusarium brachygibbosum in China.

Tobacco (Nicotiana tabacum L.) is an important cash crop in China, with an estimated production of 2.2 million tons every year (Berbeć and Matyka, 2020). In June 2020, a root rot disease was observed on tobacco (cv. Zhongyan 100) in four surveyed counties (Mianchi, Lushi, Duguan and Lingbao) in Sanmenxia. Diseased plants exhibited leaf chlorosis and purplish to brown vascular discoloration of stem, taproot and lateral roots. The disease incidence ranged from 15% to 40% in 11 surveyed fields, 36.7 ha in total. Twenty five diseased tissues were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Fifteen single-spore isolates were obtained from 25 diseased tissue samples. All cultures growing on PDA had white colonies with abundant aerial mycelia initially, turning into yellow to orange in the center and produced red pigmentation after seven days of growth. The 7-day-old cultures grown on carnation leaf agar (CLA) produced macroconidia that were curved with 3-5 septa, had wide central cells, slightly pointy apex, and measured 17.0-45.9 μm long×3.0-4.6 μm wide (n=50). The microconidia formed on CLA were slightly curved, ovoid with zero to two septa, measuring 5.4-15.5 μm long×2.0-3.2 μm wide (n=50). Spherical chlamydospores (7.58-13.52 μm; n=50) were terminal or intercalary, single or in chains. Such characteristics were typical of Fuarium brachygibbosum (Tirado-Ramírez et al. 2018). DNA from one representative single-spore isolate (MC1) was extracted, and the translation elongation factor 1-alpha (EF1-α), RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes were amplified with primers EF1/EF2, F5/G2R and RPB2F/R respectively (O'Donnell et al. 1998, 2010), and sequenced. Sequences were submitted to GenBank under accession numbers MT947796 (EF1-α), MW679536 (RPB1) and MW430664 (RPB2). The consensus sequences showed 99.70%, 99.94% and 100% identity to the sequences of F. brachygibbosum strain NRRL 34033 (accession no. GQ505418.1, HM347172.1 and GQ505482.1, Wang et al 2021). Morphological and molecular results confirmed this species as F. brachygibbosum (Al-Mahmooli, et al., 2013, Rentería -Martínez, et al., 2018). Pathogenicity tests were performed on tobacco seedlings grown on autoclaved tobacco specific substrate (Tobacco specific matrix, Ainong Biotechnology Co. Ltd, China). Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with MC1 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7-14 days after inoculation. The pathogen was reisolated from the necrotic tissue from all inoculated seedlings and was identified by sequencing partial EF1-α and RPB2 genes. Control plants remained asymptomatic and no pathogen was recovered from the control plants. Fusarium brachygibbosum is known as a pathogen of grains and cash crops in China (Shan, et al., 2017, Xia, et al., 2018). To our knowledge, this is the first report of F. brachygibbosum causing root rot on tobacco. We believe that our results will help to better understand rhizome fungal diseases affecting tobacco production in China. Acknowledgements: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010).

First Report of Phytopythium litorale Causing Crown and Root Rot on Rhododendron pulchrum in China.

During a survey of pathogenic oomycetes in Nanjing, China from June 2019 to October 2020, at least ten adjacent Rhododendron pulchrum plants at a Jiangjun Mountain scenic spot showed symptoms of blight, and crown and root discoloration . Symptomatic root tissues collected from three 6-year-old plants were rinsed with water, cut into 10-mm pieces, surface sterilized with 70% ethanol for 1 min, and plated onto 10% clarified V8 PARP agar (cV8A-PARP) containing pimaricin (20mg/liter), ampicillin (125mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20mg/liter). Four Pythium-like isolates were recovered after three days of incubation at 26°C, and purified using hyphal-tipping. Ten agar plugs (2×2 mm2) of each isolate were grown in 10 mL of 10% clarified V8 juice (cV8) in a 10 cm plate at 26°C for 3 days to produce mycelial mats, and then the cV8 was replaced with sterile water. To stimulate sporangial production, three to five drops of soil extract solution were added to each plate. Sporangia were terminal, ovoid to globose, and the size is 24 to 45.6 (mean 34.7) (n=10.8) in length x 23.6 to 36.0 (mean 29.8) (n=6.2) in width. Gametangia were not observed in cV8A or liquid media after 30 days. For colony morphology, the isolates were sub-cultured onto three solid microbial media (cV8A-PARP, potato dextrose agar, corn meal agar) . All isolates had identical morphological features in the three media. Complete ITS and partial LSU and cox2 gene regions were amplified using primer pairs ITS1/ITS4, NL1/NL4, and FM58/FM66 , respectively. The ITS, LSU, and cox2 sequences of isolate PC-dj1 (GenBank Acc. No. MW205746, MW208002, MW208003) were 100.00% (936/936 nt), 100.00% (772/772 nt), and 99.64% (554/556 nt) identical to those of JX985743, MT042003, and GU133521, respectively. We built a maximum-likelihood tree of Phytopythium species using the concatenated dataset (ITS, LSU, cox2) to observe interspecific differences. Based on the morphological characters and sequences, isolate PC-djl was identified as Phytopythium litorale . As the four isolates (PC-dj1, PC-dj2, PC-dj3 and PC-dj4) tested had identical morphological characters and molecular marker sequences, the pathogenicity of the representative isolate, PC-dj1, was tested using two inoculation methods on ten one-year-old R. pulchrum plants. For the first inoculation method, plants were removed from the pot, and their roots were rinsed with tap water to remove the soil. Each of these plants was placed in a glass flask containing 250 mL of sterile water and 10 blocks (10 x 10 mm2) of mycelial mats harvested from a three-day-old culture of P. litorale, while the other plant was placed in sterile water as a control, and incubated at 26°C. After three days, symptoms including crown rot, root rot and blight was observed on the inoculated plants whereas the control remained asymptomatic. For the second inoculation method, ten plants were dug up to expose the root ball. Ten three-day-old cV8A plugs (5×5 mm2) from a PC-dj1 culture or sterile cV8A plugs were evenly insert into the root ball of a plant before it was planted back into the original pots. Both plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 14 to 21 days, the inoculated plant had symptoms resembling those in the field , while the control plant remained asymptomatic. Each inoculation method was repeated at triplicate and the outcomes were identical. Phytopythium isolates with morphological features and sequences identical to those of PC-dj1 were recovered from rotted crown and root tissues of all inoculated plants. Previously, P. litorale was found causing diseases of apple and Platanus orientalis in Turkey, fruit rot and seedling damping-off of yellow squash in southern Georgia, USA. This is the first report of this species causing crown and root rot on R. pulchrum, an important ornamental plant species in China. Additional surveys are ongoing to determine the distribution of P. litorale in the city of Nanjing.

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan.

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O'Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch's postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

Effects of Spore Density and Interaction With Heterodera glycines on Soybean Root Rot Caused by Fusarium solani and F. tricinctum.

Fusarium root rot, caused by Fusarium solani and F. tricinctum, is a major soybean disease in the North Central United States. This study investigated the effects of the macroconidia density and the additive effects of soybean cyst nematode (SCN), Heterodera glycines, on the severity of Fusarium root rot. To determine the effect of spore density on severity, experiments were conducted in La Prairie silt loam soil in a greenhouse using conidial suspensions ranging from 101 to 106 macroconidia/ml soil. Root discoloration and lesion lengths on taproots increased as spore numbers increased, with significant effects of spore densities starting at 104 and 105 macroconidia/ml soil for F. solani and F. tricinctum, respectively. A nonlinear sigmoid model was fitted to root discoloration against density, whereas a linear regression model was fitted to root lesion length against density. The interaction between the nematode at different egg densities with the two Fusarium spp. at 105 macroconidia/ml soil was investigated. In the greenhouse, root discoloration and lesion length were significantly greater in plants inoculated with Fusarium spp. and H. glycines at 10 eggs/ml soil or greater, compared with Fusarium spp. alone. In field trials, coinfestation of soil with the two Fusarium spp. and H. glycines significantly increased root rot severity at an egg density of 16.7 eggs/ml soil. The results indicated that the presence of SCN can increase severity of root rot caused by F. solani and F. tricinctum and egg density in the soil is an important factor in the interaction.

Tooth discoloration induced by apical plugs with hydraulic calcium silicate-based cements in teeth with open apices\u2014a 2-year in vitro study

ObjectivesTo assess tooth discoloration induced by different hydraulic calcium silicate-based cements (HCSCs), including effects of blood and placement method.Materials and methodsEighty bovine teeth cut to a length of 18 mm (crown 8 mm, root 10 mm) were randomly assigned to 10 groups (n = 8), receiving orthograde apical plug treatment (APT). Apical plugs were 4 mm in length and made of ProRoot MTA (Dentsply), Medcem MTA (Medcem), TotalFill BC RRM Fast Set Putty (Brasseler), or Medcem Medical Portland Cement (Medcem) plus bismuth oxide (Bi2O3) with and without bovine blood. Further, orthograde (with or without preoperative adhesive coronal dentin sealing) and retrograde APT were compared. Teeth were obturated with gutta-percha and sealer, sealed with composite and stored in distilled water. Tooth color was measured on apical plug, gutta-percha/sealer, and crown surface before treatment versus 24 h, 1, 3, 6, 12, and 24 months after treatment by spectrophotometry. Color difference (ΔE) values were calculated and analyzed by Shapiro–Wilk test, ANOVA with post hoc tests, Friedman test, t test, and post hoc tests with Bonferroni correction (α = .05).ResultsTooth discoloration occurred in all groups with no significant differences between HCSCs (p > .05). After 24 months, color changes were prominent on roots but insignificant on crowns. Blood contamination induced a significantly decreased luminescence (p < .05). Blood had a stronger impact on tooth color than Bi2O3. No relevant effects of retrograde placement (p > .05) or preoperative dentin sealing (p > .05) were detected.ConclusionsApical plugs of the tested HCSCs cause discoloration of bovine roots, but not discoloration of bovine tooth crowns within a 24-month period.Clinical relevanceAPT should be performed carefully while avoiding direct contact with the coronal dentin, and in that case no aesthetic impairments occur.

Gaeumannomyces nanograminis, sp. nov., a hyphopodiate fungus identified from diseased roots of ultradwarf bermudagrass in the United States

ABSTRACT The genus Gaeumannomyces (Magnaporthaceae, Magnaporthales, Sordariomycetes, Ascomycota) includes root-infecting pathogens, saprobes, and endophytes. Morphological, biological, and phylogenetic analyses were employed to identify fungal isolates derived from turfgrass roots colonized with ectotrophic, dark runner hyphae. Phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes were obtained via maximum likelihood and Bayesian methods. Our isolates consistently formed a distinct and highly supported clade within Gaeumannomyces. Common and distinctive biological and morphological characters reinforced these findings. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davey), its native host, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Gaeumannomyces nanograminis, associated with rotted roots of ultradwarf bermudagrass.

Transcriptome and metabolome profiling to elucidate mechanisms underlying the blue discoloration of radish roots during storage

The internal blue discoloration of radish roots (Raphanus sativus) during storage affects their quality. We here performed transcriptome and metabolome profiling to investigate the mechanisms underlying the bluing of radish roots during storage. On comparing white radish (WR) and blue radish (BR), we identified 14,171 differentially expressed genes (upregulated: 7,383, downregulated: 6,788) and 145 differentially accumulated metabolites (upregulated: 117, downregulated: 28). Functional annotation analysis and metabolome profiling revealed that the blue discoloration of radish roots was promoted by high content of glucosinolates, oxidation system (ROS, CAT, POD) or low reduction system (GSH, GPX, APX, GST, ASA). Our results provide new insights into the underlying metabolic causes of the blue discoloration of radish roots and report candidate genes and metabolites involved in blue compound biosynthesis.

First report of Pythium ultimum causing crown rot in greenhouse grown Cannabis sativa (L.) in California.

In April of 2020 cuttings of Cannabis sativa (L.) in a greenhouse in San Mateo County, CA were observed collapsing, and further observation revealed: water-soaked stems, tan discoloration to the cortex, and discolored roots. The greenhouse irrigation system was supplied by a local stream. We collected one-liter water samples from: intake pond, reservoir tank, irrigation lines, and local potable water tap. Water samples were filtered and plated as described previously (Rollins et al., 2016). Filter papers were removed after 24 hours. Crown sections from four symptomatic plants and one asymptomatic plant were surfaced sterilized in 10% bleach for five minutes, rinsed in sterile deionized water, cut into four-millimeter long sections, and plated onto V8 media, then incubated at room temperature for three days. White mycelial growth was observed from foci within the print of the filter paper from all irrigation water samples but not the potable water supply sample. Similar mycelial growth was observed from plated crown tissue from symptomatic plants only. Observation under light microscope revealed characteristics congruent with P. ultimum, including aseptate hyphae and globose sporangia (Watanabe, 2002). Mycelia was collected for DNA extraction from each of the water and plant sample plates with DNA extractions performed using Quick DNA Fungi/Bacterial Kit (Zymo Research Irvine, CA, USA) and PCR amplified using primers ITS100/ITS4 as described by Riit et al. (2016). All amplicons were Sanger sequenced, aligned using SnapGene software (from GSL Biotech; available at snapgene.com), and compared to barcode referencPe sequences to identify the species using the BarCode of Life Database (BOLDsystems) within the National Center for Biotechnology Information nucleotide database. After trimming and aligning, all amplicons were found to be identical, yielding the 810-nucleotide long consensus ITS amplicon (accession MW114807), which aligned with Pythium ultimum ITS sequences (e.g., accession HQ643886.1) with 100% identity and homology. We then completed Koch's postulates by using pure cultures from root sections of P. ultimum to stem inoculate C. sativa plants. We used a three-millimeter corer to remove a disc of epidermis and applied a plug of pure culture to the wound. We inoculated 10 plants, with two plants mock-inoculated using clean V8 agar. Inoculation sites were wrapped in parafilm, and plants were grown in the greenhouse for 20 days. Stems of mock and oomycete inoculated plants were examined for callus formation and 30 centimeters of stem were excised from each plant. The mock inoculated plants had fully callused inoculation sites and were discolored only where wounded. P. ultimum inoculated plant inoculation sites were partially callused over and had tan discoloration of the cortex that extended 6.0 mm +/- 2.0 mm above and below the inoculation site. Stem segments above and below inoculation sites were surface sterilized and plated on V8 media as previously described and P. ultimum recovered from inoculated plants, confirmed as identical to the inoculum by ITS amplification and sequencing. Mock inoculated plant stem cultures yielded no oomycete growth. Together, these results indicate that P. ultimum has the ability to cause crown rot in C. sativa in greenhouse cultivation.