Crown Rot Symptoms Research Articles

Uncovering the fungus responsible for stem and root rot of false indigo: pathogen identification, new disease description, and genome analyses.

Calonectria spp. can cause destructive diseases on forestry crops, legumes like soybean and peanut, and ornamentals. Species of Calonectria affecting ornamental plants are not well characterized or understood, though they have been widely documented as an issue in the ornamental industry. This research focused on the molecular identification, pathogenicity validation, and genome analysis of a Calonectria sp. isolate recovered from ornamental blue false indigo (Baptisia australis) plants showing disease symptoms of crown and root rot in a commercial nursery in Virginia. The fungus on B. australis was identified as C. fujianensis (Nectriaceae, Hypocreales), a member of the C. colhuonii species complex using multilocus sequencing. Pathogenicity tests were fulfilled by inoculating C. fujianensis conidia on B. australis seedlings, confirming a causal relationship between this pathogen and the disease symptoms observed. A 62.7 Mb high-quality hybrid genome assembly generated using Illumina and Nanopore data was obtained, contained in 16 contigs, four of which were complete chromosomes. A total of 750 effectors were found in the genome, similar to cutinase and pectinase virulence factors described from other Calonectria species' genomes. Characterization of this novel disease of B. australis advances our understanding of Calonectria as an important but poorly studied group of plant pathogens.

Characterisation of Fusarium and Neocosmospora Species Associated With Crown Rot and Wilt of African Eggplant (Solanum aethiopicum) in Ghana

ABSTRACTAfrican eggplant (AEP) (Solanum aethiopicum group Gilo) is an important vegetable with considerable economic value in Ghana and tropical Africa. However, fungal diseases threaten its cultivation. Surveys conducted in 2021 and 2022 growing seasons across 35 commercial farms in five regions of Ghana revealed symptoms of crown rot and wilt affecting AEP. This study was undertaken to identify and characterise 36 fungal isolates causing these diseases in AEPs using morphological, molecular and pathogenicity assays. Morphological and molecular analyses of the Btub2, Tef‐1α and Rpb2 sequences identified two Fusarium species (F. elaeidis and F. fredkrugeri) and three Neocosmospora species (N. falciforme, N. suttoniana and N. solani) associated with the plant diseases. F. elaeidis (14 isolates) and N. falciforme (14) were the most commonly isolated species from symptomatic plants. Specifically, F. elaeidis was found in wilting plants, while F. fredkrugeri and the three Neocosmospora spp. were more associated with wilting plants with crown rot symptoms than plants with only wilt symptoms. All identified species exhibited pathogenicity when inoculated onto AEP roots and stems, confirming field observations. F. elaeidis was the most aggressive in inducing wilt symptoms, while N. solani and N. suttoniana were particularly aggressive in inducing crown rot symptoms. This study is the first to document that F. elaeidis, F. fredkrugeri, N. falciforme and N. suttoniana are pathogens causing wilt and crown rot in AEP in Ghana. These findings provide essential insights for developing effective disease management strategies to reduce losses from these fungal species.

Fusarium pseudograminearum biomass and toxin accumulation in wheat tissues with and without Fusarium crown rot symptoms.

Fusarium crown rot (FCR) is an important and devastating disease of wheat (Triticum aestivum) caused by the fungus Fusarium pseudograminearum and related pathogens. Using two distinct susceptible cultivars, we investigated the isolation frequencies of F. pseudograminearum and quantified its biomass accumulation and the levels of the associated toxins deoxynivalenol (DON) and DON-3-glucoside (D3G) in inoculated field-grown wheat plants. We detected F. pseudograminearum in stem, peduncle, rachis, and husk tissues, but not in grains, whereas DON and D3G accumulated in stem, rachis, husk, and grain tissues. Disease severity was positively correlated with the frequency of pathogen isolation, F. pseudograminearum biomass, and mycotoxin levels. The amount of F. pseudograminearum biomass and mycotoxin contents in asymptomatic tissue of diseased plants were associated with the distance of the tissue from the diseased internode and the disease severity of the plant. Thus, apparently healthy tissue may harbor F. pseudograminearum and contain associated mycotoxins. This research helps clarify the relationship between F. pseudograminearum occurrence, F. pseudograminearum biomass, and mycotoxin accumulation in tissues of susceptible wheat cultivars with or without disease symptoms, providing information that can lead to more effective control measures.

First Report of Fusarium culmorum Causing Crown Rot on Wheat in Jordan

First Report of <i>Fusarium culmorum</i> Causing Crown Rot on Wheat in Jordan

Characterization of Coniella granati Isolates Causing Pomegranate Decline in Italy.

Coniella granati, the causal agent of pomegranate crown rot, twig blight, and fruit decay, is an emerging worldwide pathogen with a heavy impact on pomegranate cultivation. In this study, we report the rapid spread of the fungus in Italian pomegranate orchards associated with crown rot symptoms and provide new results on fungal development, baseline sensitivity to different fungicides, and intraspecific variability by analyzing 11 isolates, representative of populations of the pathogen from comparable pomegranate orchards in different regions of Italy. In vitro assays showed that 25 to 30°C was the optimal range for both colony growth and conidial germination, corroborating the results previously obtained for Californian and Greek isolates. According to the baseline sensitivity assay on the response of colony growth and conidial germination to 10 fungicides, fludioxonil, thiophanate-methyl, tebuconazole, and cyprodinil were the most effective. Random amplified polymorphic DNA (RAPD) analysis, carried out using fourteen 10-mer primers, showed very low intraspecific variability (similarity coefficient >0.95), probably as a result of the recent spread of the pathogen in Italy and the uncommon occurrence of the sexual process as a source of genetic variability. In summary, this study provides new knowledge on C. granati that will be helpful for improving pomegranate crown rot management.

First Report of Vascular Wilt on Peanut in Mexico Caused by Fusarium incarnatum.

During the fall of 2020, wilt symptoms were presented in a commercial peanut field (Arachis hypogaea L.), variety 'CHAMPS' in Buenavista de Benito Juárez, México (18.460501 N, 98.627100 W). A peanut field was scout 80 days after planting, and plants presenting symptoms of root and crown rot, vascular chlorosis, and tissue death, were sampled. Disease incidence was estimated at 55% of the field. Isolations of the pathogen were made from stem and root tissues. These samples were disinfected by immersion in 1% sodium hypochlorite (NaClO) for 3 min and immersion in 70% ethanol for 1 min with 3 rinses with sterile distilled water. Subsequently, 0.5 cm fragments were removed and placed on media of potato dextrose agar (PDA) and Rose Bengal. Petri dishes were incubated in complete darkness at 26 °C for 7 days. Abundant aerial white mycelium was observed, which turned tan to brown and showed a slightly orange color on the back of the plate. Finally, pure cultures were obtained by single sporing (Aslam et al. 2020). Colonies identified as Fusarium spp. (Leslie and Summerell, 2006) were sub cultured on PDA agar media and Spezieller Nährstoffarmer (Pérez-Vázquez et al. 2022) to observe microscopic characteristics of ten isolates. Colonies of a representative strain (MA-PET-03) produced hyaline septate hyphae, macroconidia dorsoventrally curved, tapering towards both ends of 51-57 × 4.6-5.4 μm (n = 80) with most having 7 septa. Microconidia were unicellular, nonseptate, hyaline, and ovoid, 12.4-20.6 × 3.6-4.1 μm (n = 80). Chlamydospores were abundant and globose 5-11 μm diam (n = 80), intercalary, and solitary in short chains (Figure 1). The observed microscopic characteristics correspond to the description of Fusarium incarnatum (Khoa et al. 2006; Leslie and Summerell, 2006; Xia et al. 2019). The molecular analyses were done with genomic DNA extracted as previously reported by Pérez et al. (2022). A region from the translation elongation factor gene was PCR amplified using EF688/EF1251 primers (Alves et al. 2008) and from the calmodulin (CMDA) gene, using CALDF1 and CALDR1 primers (Noel et al. 2022). The corresponding PCR products were purified with the Gen Elute™ PCR Clean-Up Kit from Sigma-Aldrich Co. (St. Louis Mo. USA) and sequenced at Macrogen Inc. (Seoul, South Korea). The phylogenetic analysis was inferred using the Bayesian Inference method with 1 million generations, final standard deviation was 0.008516. The nucleotide substitution model for Calmodulin (CMDA) was GTR + G and for TEF1 GTR + I + G. This analysis showed that strain MA-PET-03 shared 100% identity (Figure 2) with F. incarnatum ex-type strain CBS 132.73 (CMDA: MN170342; TEF1: JMN1704761) from Pointed gourd (Trichosanthes dioica) in Malawi Africa. The sequences of strain MA-PET-03 were deposited in GenBank (CMDA: OQ679820; TEF1: OQ679821). The pathogenicity tests were carried out with a total of 20 peanut plants, variety 'CHAMPS', 18 days after having been sown in groups of five seeds in 250 g plastic pots, containing a sterilized mixture of Peatmoss and Agrellite (1:1 v./v), with four repetitions. The seeds were inoculated by immersion in 20 mL of spore suspension (106 conidia/mL) isolated from F. incarnatum for 10 min. The plants were maintained in a greenhouse (70% relative humidity and 28 °C) until the appearance of disease symptoms of. Likewise, 10 control plants were inoculated with sterile water. The experiment was repeated twice. The symptoms developed 15 days after inoculation, the plants presented symptoms of chlorosis, wilting of leaves, stems, and roots, a manifestation similar to that observed in the field, while the control plants remained healthy. F. incarnatum was consistently reisolated from inoculated stems and roots and identified by the microscopic characteristics described above. Peanut leaf blight and wilt disease caused by F. incarnatum has been reported in India (Thirumalaisamy et al. 2019). This first report emphasizes that this phytopathogen is a new threat for peanut producers in Mexico, which is why our finding suggests the need to seek new strategies for its control.

Flagella-Driven Motility Is Critical to the Virulence of Xanthomonas fragariae in Strawberry.

Xanthomonas fragariae (X. fragariae) is the causal agent of angular leaf spots (ALS) in strawberry plants. Recently, a study in China isolated X. fragariae strain YL19, which was observed to cause both typical ALS symptoms and dry cavity rot in strawberry crown tissue; this was the first X. fragariae strain to have both these effects in strawberry. In this study, from 2020 to 2022, we isolated 39 X. fragariae strains from diseased strawberries in different production areas in China. Multilocus sequence typing (MLST) and phylogenetic analysis showed that X. fragariae strain YLX21 was genetically different from YL19 and other strains. Tests indicated that YLX21 and YL19 had different pathogenicities toward strawberry leaves and stem crowns. YLX21 did not cause ALS symptoms, rarely caused dry cavity rot in strawberry crown after wound inoculation, and never caused dry cavity rot after spray inoculation, but it did cause severe ALS symptoms after spray inoculation. However, YL19 caused more severe symptoms in strawberry crowns under both conditions. Moreover, YL19 had a single polar flagellum, while YLX21 had no flagellum. Motility and chemotaxis assays showed that YLX21 had weaker motility than YL19, which may explain why YLX21 tended to multiply in situ within the strawberry leaf rather than migrate to other tissues, causing more severe ALS symptoms and mild crown rot symptoms. Taken together, the new strain YLX21 helped us reveal critical factors underlying the pathogenicity of X. fragariae and the mechanism by which dry cavity rot in strawberry crowns forms.

Identification and Chemical and Biological Management of Fusarium Root and Crown Rot Disease of Oakleaf Hydrangea.

Oakleaf hydrangea (Hydrangea quercifolia) is an important ornamental plant grown in Tennessee. In May 2018, after late spring frost, cultivars Pee Wee and Queen of Hearts showed root and crown rot symptoms and identification and management of the disease was a major concern. The objective of this research was to identify the causal organism of this disease and develop management recommendations for nursery growers. Isolates from the infected root and crown parts were subjected to microscopy and the morphology of fungi resembled Fusarium. Molecular analysis was conducted by amplifying the internal transcribed spacer (ITS) of ribosomal DNA, beta-tubulin (b-Tub), and translation elongation factors 1-α (EF-1α) regions. Fusarium oxysporum was identified as a causal organism based on morphological and molecular analysis. Pathogenicity test was done to complete the Koch's postulates by drenching containerized oakleaf hydrangea with conidial suspension. Experiments were conducted to evaluate different chemical fungicides and biological products with different rates for Fusarium root and crown rot management in container grown 'Queen of Hearts'. Plants were inoculated by drenching containerized oakleaf hydrangea with 150 mL conidial suspension of F. oxysporum maintaining the concentration of 1×106 conidia/mL. Root and crown rot were assessed using a scale of 0-100%. Recovery of F. oxysporum was recorded by plating root and crown sections. Chemical fungicides such as mefentrifluconazole (BAS75002F), the low rate (1.09 mL/L) of difenoconazole + pydiflumetofen (Postiva), and the high rate (1.32 mL/L) of isofetamid (Astun) and biopesticide: the high rate (1.64 g/L) of ningnanmycin (SP2700 WP) effectively reduced Fusarium root rot severity and pyraclostrobin effectively reduced Fusarium crown rot severity in both trials.

Establishment of the Recombinase Polymerase Amplification-Lateral Flow Dipstick Detection Technique for Fusarium oxysporum.

Fusarium oxysporum causes crown rot, wilt, root rot, and many other major plant diseases worldwide. During the progression of strawberry crown rot disease, the pathogen is transmitted from the mother plant to the seedling through the stolon, with obvious characteristics of latent infection. Therefore, rapid and timely detection of F. oxysporum is important for efficient disease management. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection technique was developed for the rapid detection of F. oxysporum on strawberry plants by targeting the CYP51C gene, which is unique to Fusarium spp. Because this RPA-LFD detection technique was highly specific to F. oxysporum, other Fusarium and non-Fusarium fungi were not detected. The optimal reaction temperature and time for this technique were 39°C and 8 min, respectively. The detection limit was 1 pg of F. oxysporum genomic DNA in a 50-μl reaction system. A total of 46 strawberry plants with or without crown rot symptoms collected from Jiande, Changxing, and Haining in Zhejiang Province were further assessed for F. oxysporum infection using both RPA-LFD and traditional tissue isolation techniques. The RPA-LFD test showed that 32 of the 46 strawberry plants tested were positive for F. oxysporum, while in the traditional isolation technique, F. oxysporum was isolated from 30 of the 46 strawberry plants. These results suggest that our established RPA-LFD method is rapid, sensitive, and highly specific in detecting F. oxysporum infection in strawberry plants.

First report of Lasiodiplodia brasiliensis causing crown rot on banana in Brazil.

Postharvest diseases compromise banana quality and cause high economic losses in Brazil. Among them, the crown rot prevails and its causal agents belong to distinct fungal species such as Colletotrichum musae (Berk. & Curt.) von Arx, Fusarium spp., and Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (Griffee and Burden 1976; Ploetz et al. 2003). Symptoms of crown rot were observed on banana fruits of cv. Williams in a commercial area in Assu, Rio Grande do Norte, Brazil (04°54'0.06"S, 37°22'6.02"W) in 2017. The samples were collected, superficially disinfected with NaClO (2%), and incubated in a wet chamber at 25 °C, with a 12 h photoperiod, for approximately 3 days. After the appearance of disease symptoms and pathogen signs, mycelia were transferred from the lesions to obtain pure cultures on a potato dextrose agar (PDA) medium. Thus, a monosporic culture was obtained (isolate BAN82). The fungus produced pycnidia with conidia on potato carrot agar (PCA) culture medium containing pine needles, after four weeks of incubation at 28 °C. The conidia were hyaline when immature and brown with central transverse septum when mature. The presence of conidiogenous cells, paraphyses, and conidiophores also were observed. The conidia present ovoid format measuring 20-28 x 11-14 µm (n=50). The fungal colony produced abundant aerial mycelia of mouse grey coloration, progressing to dark mouse grey (Rayner 1970), on PDA for 15 days to 28 °C. The growth rate was 29.3 mm/day on PDA. The genomic DNA was extracted and amplified PCR with primers TEF1-688F/TEF1-1251R, ITS1/ITS4, and Bt2a/Bt2b and sequenced in both directions. The TEF1 and TUB2 sequences showed 100%, and the ITS showed 93.06% identity with the sequences of Lasiodiplodia brasiliensis (GenBank accession numbers: ON623895, TEF1, ON623896, TUB2, and ON599012, ITS. Multiple alignments of the combined dataset of the isolate and representative sequences obtained from GenBank were submitted phylogenetic analyses to bayesian inference (IB) with posterior probabilities of 10,000,000 generations. The morphological characteristics together with multigenic analysis of the three genomic regions made it possible to identify the BAN82 isolate as Lasiodiplodia brasiliensis, showing bootstrap support of posterior probabilities of 0,98 in the IB analysis. The pathogenicity was evaluated on 16 banana fruits from cv. Prata Catarina, at the point of harvest. For inoculation, the bananas were disinfected with water, soap, and, NaClO (2%). Posteriorly, the fruits were wounded on both ends, followed by the deposition of 5mm diameter mycelial plugs from the fungal culture, within 7 days of the growth. After the inoculation, the fruits were incubated in plastic boxes in a wet chamber at 25 °C, with 12 h photoperiod, for 3 days. To complete Koch's postulates, the isolate was inoculated again into 16 other banana fruits from cv. Prata Catarina. The negative control fruits were not inoculated with the pathogen, only with PDA discs. The BAN82 isolate was pathogenic to the banana cv. Prata Catarina. In the Brazilian Northeast, L. brasiliensis was described in 2014 as being associated with papaya stem rot. Up to the moment, there are no reports of L. brasiliensis as the causal agent of crown rot on bananas from Brazil (Netto et al. 2014; Farr and Rossman 2022). Thus, our work is the first to report L. brasiliensis causing crown rot on banana fruits cv. Prata Catarina in Brazil.

Anastomosis Groups and Mycovirome of Rhizoctonia Isolates Causing Sugar Beet Root and Crown Rot and Their Sensitivity to Flutolanil, Thifluzamide, and Pencycuron

Anastomosis groups (AGs) or subgroups of 244 Rhizoctonia isolates recovered from sugar beet roots with symptoms of root and crown rot were characterized to be AG-A, AG-K, AG-2-2IIIB, AG-2-2IV, AG-3 PT, AG-4HGI, AG-4HGII, and AG-4HGIII, with AG-4HGI (108 isolates, 44.26%) and AG-2-2IIIB (107 isolates, 43.85%) being predominate. Four unclassified mycoviruses and one hundred and one putative mycoviruses belonging to six families, namely Mitoviridae (60.00%), Narnaviridae (18.10%), Partitiviridae (7.62%), Benyviridae (4.76%), Hypoviridae (3.81%), and Botourmiaviridae (1.90%), were found to be present in these 244 Rhizoctonia isolates, most of which (88.57%) contained positive single-stranded RNA genome. The 244 Rhizoctonia isolates were all sensitive to flutolanil and thifluzamide, with average median effective concentration (EC50) value of 0.3199 ± 0.0149 μg·mL-1 and 0.1081 ± 0.0044 μg·mL-1, respectively. Among the 244 isolates, except for 20 Rhizoctonia isolates (seven isolates of AG-A and AG-K, one isolate of AG-4HGI, and 12 isolates of AG-4HGII), 117 isolates of AG-2-2IIIB, AG-2-2IV, AG-3 PT, and AG-4HGIII, 107 isolates of AG-4HGI, and six isolates of AG-4HGII were sensitive to pencycuron, with average EC50 value of 0.0339 ± 0.0012 μg·mL-1. Correlation index (ρ) of cross-resistance level between flutolanil and thifluzamide, flutolanil and pencycuron, and thifluzamide and pencycuron was 0.398, 0.315, and 0.125, respectively. This is the first detailed study on AG identification, mycovirome analysis, and sensitivity to flutolanil, thifluzamide, and pencycuron of Rhizoctonia isolates associated with sugar beet root and crown rot.

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.

Host Susceptibility of CIMMYT’s International Spring Wheat Lines to Crown and Root Rot Caused by Fusarium culmorum and F. pseudograminearum

The destructive soilborne Fusarium species is one of the most serious challenges facing agriculture. Mycotoxins produced by Fusarium spp. can induce both acute and chronic toxic effects on humans and animals. Massive investments have been made in the last few decades to develop an appropriate management strategy to control Fusarium species in cereals, particularly in wheat, using genetic resistance and other practices, with varied outcomes. The purpose of this research was to find new sources of resistance to both Fusarium culmorum and F. pseudograminearum, which are wheat’s most destructive pathogens in seedlings and adult plants stages. In this study, 26 lines were selected and promoted from a total of 200 spring wheat germplasm received from CIMMYT Mexico plus 6 local check lines. The 32 lines were screened for their resistance reactions to both Fusarium species under different environmental conditions. The discriminant factorial analysis indicated that 7, 12, and 5 were resistant lines against F. culmorum under field, greenhouse, and growth room conditions, respectively. Four lines, L12, L19, L21, and L26, were found to be jointly resistant at the adult and seedling stages in the field and greenhouse. On the other hand, only moderately resistant lines were found for F. pseudograminearum but not completely resistant, which was limited to growth room conditions. Interestingly, five lines (L10, L13, L17, L25, and L28) have shown resistant properties to both Fusarium species. To further evaluate the yield performance of the best-selected 26 lines plus 6 check lines, field trials were conducted under ± F. culmorum inoculum. The highest yield values were obtained from three check lines, as well as the L26, which showed consistency in its reaction to F. culmorum under both field and greenhouse conditions, and produced a high yield (5342 kg/ha). Based on the result obtained, L26 showed a high potential to improve wheat yield and resistance to F. culmorum-caused root and crown rot; therefore, it should be used in wheat crossing programs. Having Fusarium-resistant varieties will ultimately reduce crown rot symptoms and increase grain quality by reducing mycotoxin levels.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 μm, n = 50) and occasionally one-septate (21.89 ± 2.01 μm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 μm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China.

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch's postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

Identity and Pathogenicity of Fungi Associated with Root, Crown, and Vascular Symptoms Related to Winter Squash Yield Decline.

Winter squash (Cucurbita maxima cultivar Golden Delicious) produced in Oregon's Willamette Valley for edible seed production has experienced significant yield losses because of a soilborne disease. The symptoms associated with this disease problem include root rot, crown rot, and vascular discoloration in the stems, leading to a severe late season wilt and plant collapse. Through field surveys, Fusarium oxysporum, F. solani, F. culmorum-like fungi, Plectosphaerella cucumerina, and Setophoma terrestris were identified to be associated with diseased tissues, and each produced symptoms of root rot, crown rot, or stem discoloration in preliminary pathogenicity trials. In this study, 219 isolates of these species were characterized by molecular identity analyses using BLAST of the internal transcribed spacer and translation elongation factor 1 alpha genomic regions and by pathogenicity testing in outdoor, large-container trials. Molecular identity analyses confirmed the identity of isolates at 99 to 100% similarity to reference isolates in the database. In pathogenicity experiments, F. solani produced the most severe symptoms, followed by F. culmorum-like fungi, F. oxysporum, P. cucumerina, and S. terrestris. Some treatments of mixed-species inoculum produced symptom severity greater than what was expected from individual species. In particular, the mixture of F. culmorum-like fungi, F. oxysporum, and P. cucumerina and the mixture of F. culmorum-like fungi, F. solani, and S. terrestris had symptom ratings as high as that of F. solani by itself. Results indicate that this soilborne disease is caused primarily by Fusarium solani, but interactions between the complex of F. solani, F. culmorum-like fungi, F. oxysporum, and P. cucumerina can exacerbate disease severity.

First Report of Crown Rot of Banana Caused by Fusarium proliferatum in Georgia, U.S.A.

Crown rot is one of the most damaging disease of banana fruit characterized by rot and necrosis of crown tissues. In severe cases, the disease can spread to the pedicel and banana pulp. Crown rot can be infected by several common fungi, including Lasiodiplodia theobromae, Musicillium theobromae, Colletotrichum musae, and a complex of Fusarium spp. and lead to softening and blackening of tissues (Lassois et al., 2010; Kamel et al., 2016; Triest et al., 2016; Snowdon, 1990). In November 2020, typical crown rot of banana fruits (cv. Pisang Awak, belonging to the tetraploid AABB genome) were observed from UGA Banana Research 12 Plots, Tifton, GA, with incidence rates of 15%. Initial symptoms appeared in the infected crown of green banana fruits. As the infection progressed, the crown tissues became blackened and softened, followed by an internal development of infection affecting the peduncle and the fruit, triggered early ripening of bananas. At last, the development of necrosis on the pedicels and fruits appeared and caused the fingers to fall off. To identify the pathogen, tissue pieces (~0.25 cm2) from the infected crown and pedicles were surface-sterilized in a 10% bleach solution for 1 min, followed by 30 s in 70% EtOH. The disinfected tissues were rinsed in sterile water 3 times and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 25°C in the dark for 5-10 days. Isolates of the pathogen were purified using the single-spore isolation method (Leslie and Summerell 2006). Colonies on PDA produced fluffy aerial mycelium and developed an intense purple pigment when viewed from the underside. A range of colony pigmentation and growth rates were observed among the isolates. The microconidia were ovoid, hyaline, or ellipse in shape. The morphological features of the isolates were identified as Fusarium proliferatum (Leslie and Summerell, 2006). To further identify the isolates, genomic DNA was extracted from a representative isolate. And the internal transcribed spacer (ITS) region, the partial elongation factor (TEF1-α) gene and the β-tubulin gene (TUB2)were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), EF-1 /EF-2 (O'Donnell et al. 1998) and B-tub1 /B-tub2 (O'Donnell and Cigelnik, 1997), respectively. The amplicons were sequenced and deposited in NCBI (accessions no. MZ292989, MZ293071 for ITS: MZ346602, MZ346603 for TEF1-α and MZ346600 and MZ346601 for B-tub). The ITS, TEF1-α, and B-tub sequences of the isolates showed 100% sequence similarity with Fusarium proliferatum isolates (accessions no. MT560212, LS42312, and LT575130, respectively) using BLASTn in Genbank. For pathogenicity testing, three whole bunched bananas sterilized with 10% bleach solutions and washed by sterilized water, were cut into 5 bananas per brunch. The cut surface of the banana crown was inoculated with conidial suspension (1.0 × 107 cfu/ml) of the pathogen with pipette tips. Equal number of bananas were treated with sterilized water in the same volume as a control. All bananas were sealed in a plastic bag and incubated at 25°C. After 7 days post inoculation, all inoculated bananas showed initial crown rot symptoms while no symptoms were observed on the control bananas. The fungus was re-isolated from the symptomatic tissues of infected bananas and confirmed to be genetically identical to F. proliferatum of the original inoculated strains according to morphological characteristics and molecular identification, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F. proliferatum causing crown rot on bananas in Georgia, USA.

Peronosporales Species Associated with Strawberry Crown Rot in the Czech Republic.

The symptoms of crown rot on strawberry plants are considered typical for the pathogen Phytophthora cactorum, which causes high losses of this crop. However, an unknown number of related species of pathogens of Peronosporales cause symptoms quite similar to those caused by P. cactorum. To determine their spectrum and importance, strawberry plants were sampled from 41 farms in the Czech Republic. The cultures were isolated from the symptomatic plants using the baiting method, with subsequent cultivation on a semiselective medium. Isolates were identified to the species level using nuclear ribosomal internal transcribed spacer (ITS) barcoding after preliminary morphological determination. In total, 175 isolates of 24 species of Phytophthora, Phytopythium, Pythium, and Globisporangium were detected. The most represented was Phytophthora cactorum, with 113 (65%) isolates, which was recorded in 61% of farms, and the Pythium dissotocum complex with 20 (11%) isolates, which was recorded in 27% of farms. Other species were represented in units of percent. Large differences between farms in the species spectra were ascertained. The differences between species in cardinal growth temperatures and different management of the farms are discussed as a main reason for such a diversification. Regarding the dissimilar sensitivity of various species of Peronosporales against fungicides, the proper determination of the cause of disease is of crucial significance in plant protection.

First Report of Pythium aphanidermatum Causing Crown and Root Rot on Industrial Hemp in China.

In July 2020, symptoms of crown and root rot were observed on about 10% of 4-month-old plants of industrial hemp Cannabis sativa cultivar Yunma-1 in Weifang City, Shandong Province in eastern China (Fig 1). During this month, the local temperature ranged from 19-32°C, and the total precipitation was 148mm. The disease symptoms included leaf chlorosis, crown and root rot, stunted growth, and wilting (Figs. 1 and 2). The diseased stem and root tissues were collected and cut into fragments of 0.5cm each. The fragments were surface-sterilized by dipping into 1% NaClO for 1 min, rinsed in sterile water and plated on potato dextrose agar (PDA) and on oomycetes-selective medium PARP (Jeffers and Martin 1986). The plates were incubated at 25°C in the dark for 3 days and 18 total single-hyphal purified isolates were obtained for further analyses with 8 from PDA and 10 from PARP. The colonies of all 18 isolates were white, had abundant aerial hyphae, and were cottony in appearance, resembling Pythium spp (Watanabe 2002). The grass-leaf method (Van Der Plaats-Niterink 1981) induced their sexual reproduction. The size and shape of hyphae, oogonia, antheridia, and oospores were all consistent with those of Pythium aphanidermatum (Fig 3). DNA was extracted from three isolates and their internal transcribed spacer (ITS) regions of rDNA were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990). The ITS sequences of all three isolates were identical to each other (GenBank accession OK091124.1) and showed a 100% query coverage and 99.88% nucleotide sequence identity with that of type strain of P. aphanidermatum (GenBank accession AY598622.2). Pathogenicity tests were performed with three isolates on hemp cultivar B1. Sterile substrates were prepared in 2L-pots containing peat soil and vermiculite in a 2:1 ratio, with test hemp plants grown from rooted stem cuttings. Plants were kept in a greenhouse at 22 to 27°C under 16 h photoperiod, watered every two days (about 200ml each time) and supplied commercial nutrient solution once a week. A month after transplanting to pots, a wound of 1 mm deep and 10 mm long (made by a sterilized needle) on the surface of the root crown area of the main stem was inoculated with an 8-mm-diameter agar disk of mycelia grown on PDA for 4 days. Six plants were tested for each isolate and three plants were inoculated with sterile agar medium without mycelia as negative controls. The experiment was repeated twice. After one month, plants inoculated with P. aphanidermatum isolates showed the same disease symptoms as observed on field plants while all negative control plants remained disease-free. P. aphanidermatum was reisolated from the diseased tissue and confirmed to be identical to those inoculated based on ITS sequencing and colony morphology. To our knowledge, this is the first report of P. aphanidermatum causing crown and root rot on hemp in China. With an estimated 66,700 hectares hemp cultivation in China producing over US$1 billion worth of hemp fiber (McGrath 2020), this pathogen represents a serious threat to the hemp industry. This pathogen has been reported on hemp in the US and Canada (Beckerman et al. 2017; Punja et al. 2018). The origin of P. aphanidermatum on hemp in China and its relationship to those in North America remain to be examined.

First report of Pythium schmitthenneri on olive trees and in Morocco

In a grove in north-eastern Morocco in 2020, severe root rot and crown rot symptoms were noticed on olive trees (Olea europaea). Pythium schmitthenneri was identified as the pathogen based on conidia morphology and cytochrome oxidase subunit II (COXII) gene sequence analysis. Koch's postulates were verified by re-isolation of the fungus P. schmitthenneri from artificially inoculated olive seedlings after a pathogenicity test. This is the first time P. schmitthenneri has been identified as the cause of olive tree root and crown rot in Morocco.