Strawberry Root Rot Research Articles

Efficacy of Pseudomonas fluorescens and Organic Amendments Against Black Root Rot Disease Caused by Rhizoctonia solani in strawberry (Fragaria x ananassa Duch.)

Black root rot of strawberry caused by Rhizoctonia solani is one of the devastating soil borne disease. An experiment was conducted to evaluate the efficacy of organic amendments, PGPR (Pseudomonas Fluorescens) and botanical treatment for the management of R. solani on strawberry. The study was laid out in Randomized Block design (RBD) including 4 r eplications with 8 treatments in pot condition at the greenhouse of Department of Plant Pathology, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, Uttar Pradesh, during Rabi season 20232024. The treatments consisted of combination of FYM, vermicompost, biomix compost, neem cake, cocopeat and botanical neem leaf extract were used as soil amendments and P. fluorescens as root treatment for evaluation on the plant growth, yield, physicochemical parameters (TSS and total ascorbic acid), disease intensity (%) and benefit cost ratio of strawb erry. The present investigation results revealed that T7 – P. fluorescens (0.3%) + FYM @ 100g + biomix compost @ 15g + vermicompost @ 15g + neem leaf extract @10% had the most promising results in term of maximum plant height (14.9 cm), maximum leaf number(15.75), days taken to first flowering (86 days), berry length (4.08 cm), berry diameter (2.84 cm), yield (38.87 qha-1), TSS (8.80 °Brix), total ascorbic acid (51.60mg/100g) and B:C ratio (1:2.24). It was also observed that T7 superior over other treatments giving least per cent disease intensity (30.75 %) followed by T6 – FYM @1 00g + P. fluorescens (0.3%) + biomix compost @15g + neem cake @15g + neem leaf extract @10% (35.50%). These findings highlight the potential of organic amendments, PGPR (P. fluorescens) and botanical treatments, as effective alternatives for managing black root rot disease caused by R. solani in strawberry cultivation.

Unraveling the Interplay: Soil Biogeochemical Factors Shaping the Efficacy of Anaerobic Soil Disinfestation in Suppressing Fusarium Root Rot of Strawberry.

Nontoxic alternatives to chemical soil fumigants for suppressing soilborne pathogens such as Fusarium oxysporum (Fo), one causative agent of strawberry black root rot complex prevalent in the Southeastern United States, are urgently needed. A promising alternative is anaerobic soil disinfestation, in which soil is amended with labile organic materials, irrigated to field capacity, and tarped to induce anaerobic fermentation for a brief period before planting. Pathogen-suppression mechanisms of anaerobic soil disinfestation include anaerobic conditions and generation of reduced metal cations (Fe2+ and Mn2+) and volatile fatty acids (VFAs; e.g., acetic, n-butyric, isovaleric, and others). However, little is known about how the interaction between VFAs, reduced metals, soil texture, and liming influences suppression of Fo. We investigated Fo suppression by VFAs and reduced metal cations in both aqueous and soil-based incubation trials. Inoculum containing Fo chlamydospores was added to aqueous medium containing either 5 or 10 mmol/liter VFAs and either 0.01 or 0.05% (wt/wt) reduced metals. In soil-based incubations, chlamydospore-containing inoculum was applied to sandy, sandy loam, and silty clay soil saturated by solutions containing 10 or 20 mmol/liter VFAs with or without 0.05% (wt/wt) reduced metals. VFAs, particularly in combination with Fe2+ in aqueous solutions and Mn2+ in soils, significantly reduced Fo viability. At the same time, liming and higher soil clay content reduced the effectiveness of VFAs and reduced metals for suppressing Fo, highlighting the influence of soil pH and soil texture on anaerobic soil disinfestation effectiveness.

Synergistic Biocontrol and Growth Promotion in Strawberries by Co-Cultured Trichoderma harzianum TW21990 and Burkholderia vietnamiensis B418.

This study aimed to investigate the efficiency of the secondary metabolites (SMs) produced by a co-culture of Trichoderma harzianum TW21990 and Burkholderia vietnamiensis B418 in the control of Colletotrichum siamense CM9. A fermentation filtrate of B418 + TW21990 co-culture (BT21) produced a notable increase in the inhibition rate of CM9 compared to those of TW21990 and B418 monocultures, which reached 91.40% and 80.46% on PDA plates and strawberry leaves, respectively. The BT21 fermentation broth exhibited high control efficiency on strawberry root rot of 68.95% in a pot experiment, which was higher than that in the monocultures and fluazinam treatment. In addition, BT21 treatment promoted strawberry root development, improved antioxidative enzyme activities in the leaves and roots, and enhanced the total chlorophyll content of the strawberry leaves. UHPLC-MS/MS analysis of fermentation filtrates was performed to elucidate SM variations, revealing 478 and 795 metabolites in BT21 co-culture in positive and negative ion modes, respectively. The metabolomic profiles suggested abundant SMs with antagonistic capabilities and growth-promoting effects: 3-(propan-2-yl)-octahydropyrrolo [1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Val)), 3-[(4-hydroxyphenyl)methyl]-octahydropyrrolo[1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Tyr)), 3-indoleacetic acid (IAA), 2-hydroxycinnamic acid, 4-aminobutyric acid (GABA), bafilomycin B1, and DL-indole-3-lactic acid (ILA) were significantly enhanced in the co-culture. Overall, this study demonstrates that a co-culture strategy is efficient for inducing bioactive SMs in T. harzianum and B. vietnamiensis, which could be exploited as a novel approach for developing biocontrol consortia.

First report of root rot of strawberry caused by Fusarium cugenangense, a member of the F. oxysporum species complex, in Tennessee, USA.

Strawberry (Fragaria × ananassa Duch) in Tennessee is cultivated on plastic mulched beds annually, and production is limited primarily by multiple oomycete and fungal root rot pathogens that result in reduced vigor and black root rot disease symptoms. In early June 2018, plants (cv. Chandler) with reduced shoot vigor and size, and black, necrotic stunted roots were collected from Rhea County, TN. Roots and crowns of 10 plants were cut into 1-3 cm pieces and surface sterilized with 0.6% NaOCl, followed by 70% ethanol for 1 min each, and plated on water agar. White mycelia produced after 3 days were transferred to potato dextrose agar amended with 10 mg/liter rifampicin. After 10 days, fungal colonies were light purple on the surface and dark purple on the colony underside, later developing blue-black pigmentation on the underside. Microconidia on carnation leaf agar were ovoid to ellipsoid, aseptate or septate and 8.0 to 24.2 (13.7) × 3.0 to 4.5 (3.8) μm in size, macroconidia were 3 to 5 septate and falcate to almost straight and 33.7 to 52.8 (44.4) × 4.0 to 5.5 (4.9) μm in size (n=80); both conidia were produced on monophialides. Chlamydospores were globose and subglobose, formed terminally and intercalary on aerial, submerged, and surface mycelium, singly or in pairs and were abundantly produced in sucrose broth and on synthetic nutrient-poor agar (SNA) (diam. 7.6 μm). Morphology was consistent with Fusarium oxysporum (Leslie and Summerell, 2006) and F. cugenangense, a member of the F. oxysporum species complex, as described by Maryani et al. (2019). Fungal mycelia were used for PCR (Phire Plant Direct PCR Master Mix, Thermo Scientific, CA) and the translational elongation factor 1-α (EF1α) region was amplified with primers EF-1/EF-2 (O'Donnell et al., 1998), internal transcribed spacer (ITS) regions amplified with primers ITS1/ITS2 (White et al. 1990), and the RNA polymerase second largest subunit region (RPB2) with primer pairs 5f2/7cr and 7cf/11ar (O'Donnell et al., 2022). PCR products of isolate SC5 were sequenced, and sequences compared to all sequences in the FUSARIOID-ID database using polyphasic identification (Crous et al., 2021) with EF1α (GenBank Accession No. ON703236) and RPB2 (OR472390) sequences. The highest similarity (100%) was with isolates of F. cugenangense, including ex-type isolate InaCC F984 (99.94% similarity) (Maryani et al., 2019). F. cugenangense is closely related to F. callistephi and F. elaeidis, but both species lack chlamydospores, and F. elaeidis has polyphialides (Lombard et al, 2019). To satisfy Koch's postulates, healthy rooted strawberry plants produced in soilless media were transplanted into 4 plastic pots (1.2-liter) containing 5% (w/v) fungal inoculum (grown on barley grain) and mixed into the top 5-cm of peat-based soilless medium. Pots were incubated at 25°C and 50% RH in a growth chamber. Four pots without inoculum served as controls. The trial was repeated. Within 8 weeks, all inoculated plants had low vigor, with necrotic and stunted roots. Root sections of control and inoculated plants were plated, and the pathogen was re-isolated from diseased roots of all inoculated plants only and confirmed as F. cugenangense based on morphology and sequence analysis. To our knowledge, this is the first report of F. cugenangense, or any member of the F. oxysporum species complex, causing root rot of strawberry in Tennessee and could be an important component of the production-limiting black root rot disease complex of strawberry.

Biological Control of Root Rot of Strawberry by Bacillus amyloliquefaciens Strains CMS5 and CMR12.

Strawberry root rot caused by Fusarium solani is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional chemical fungicides. To obtain antagonistic bacteria with a high biocontrol effect on strawberry root rot, over 72 rhizosphere bacteria were isolated from the strawberry rhizosphere soil and screened for their antifungal activity against F. solani by dual culture assay. Among them, strains CMS5 and CMR12 showed the strongest inhibitory activity against F. solani (inhibition rate 57.78% and 65.93%, respectively) and exhibited broad-spectrum antifungal activity. According to the phylogenetic tree based on 16S rDNA and gyrB genes, CMS5 and CMR12 were identified as Bacillus amyloliquefaciens. Lipopeptide genes involved in surfactin, iturin, and fengycin biosynthesis were detected in the DNA genomes of CMS5 and CMR12 by PCR amplification. The genes related to the three major lipopeptide metabolites existed in the DNA genome of strains CMS5 and CMR12, and the lipopeptides could inhibit the mycelial growth of F. solani and resulted in distorted hyphae. The inhibitory rates of lipopeptides of CMS5 and CMR12 on the spore germination of F. solani were 61.00% and 42.67%, respectively. The plant-growth-promoting (PGP) traits in vitro screening showed that CMS5 and CMR12 have the ability to fix nitrogen and secreted indoleacetic acid (IAA). In the potting test, the control efficiency of CMS5, CMR12 and CMS5+CMR12 against strawberry root rot were 65.3%, 67.94% and 88.00%, respectively. Furthermore, CMS5 and CMR12 enhanced the resistance of strawberry to F. solani by increasing the activities of defense enzymes MDA, CAT and SOD. Moreover, CMS5 and CMR12 significantly promoted the growth of strawberry seedlings such as root length, seedling length and seedling fresh weight. This study revealed that B. amyloliquefaciens CMS5 and CMR12 have high potential to be used as biocontrol agents to control strawberry root rot.

First Report of Strawberry Root Rot Caused by Fusarium falciforme in China.

Root rot caused by Fusarium spp. is a destructive disease affecting agricultural regions worldwide. Strawberries (Fragaria × ananassa Duch.) are an economically important crop in China. In March 2023, root rot was observed in strawberries grown in Jinan, Shandong Province, China. Symptoms included leaf wilt, necrotic roots, and plant death (Figure 1). Four strawberry samples (two symptomatic and two asymptomatic) were collected from ~2-acre fields where the disease incidence rate ranged from 2 to 3%. Tissue pieces (5 mm × 5 mm × 5 mm) from two healthy and two diseased strawberry root tissues were surface-disinfected with 75% ethanol for 3 min, treated with 10% sodium hypochlorite for 5 min, and washed three times with sterile water. These pieces were cultured for 5 days at 28°C on potato dextrose agar (PDA) containing 200 mg/L timentin. Typical Fusarium spp. like growth was observed on plates with the two symptomatic samples. Two representative fungal isolates (CM1 and CM2) with similar morphological characteristics were purified using the single-spore method (Figure 1). CM1 showed an average growth rate of 5 mm/d in PDA and comprised of several white-to-cream aerial mycelia after 5 d. After cultivation in carnation leaf agar medium for 7 d, falciform macroconidia, with blunt apical cells and slightly hooked basal cells comprising 3 to 4 septa of varying sizes (20 to 39)×(3.6 to 6.7 μm) were observed (n=50) (Figure 1). The chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, and 3.1-10.5 μm in diameter (Figure 1). The microconidia on PDA were (5.8 to 13.6)× (2.5 to 3.3) μm in size (n=50). These morphological characteristics are consistent with previous descriptions of the Fusarium solani species complex (FSSC). DNA was extracted using the CTAB method (Stenglein and Balatti 2006). The internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified and sequenced using specific primers (O'Donnell et al. 2010). The ITS (OR526528, OR526529), tef1 (OR536947, OR536948), rpb1 (OR536949, OR536950), and rpb2 (OR536951, OR536952) sequences of the CM1 and CM2 isolates were uploaded to the NCBI database. BLASTn analysis revealed that the ITS, tef1, rpb1, and rpb2 sequences were 99.1-100% identical to those of the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978. A phylogenetic tree based on the ITS, tef1, rpb1, and rpb2 sequences was generated using MEGA v.11 via the maximum-likelihood method (Tamura et al. 2021). CM1 clustered with the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978 and belonged to the FSSC based on its morphological and molecular characteristics (Figure 2). To test for pathogenicity, the roots of nine 3-month-old healthy strawberry (cv. Akihime) plants were exposed to conidial suspensions (1×108 spores/mL) of the CM1 isolate. Another nine root samples were treated with sterile water and used as controls. All strawberry plants were maintained in a growth chamber under a 12/12 h light/dark cycle at 28°C and 90% relative humidity and the experiment was repeated three times. After one month, the inoculated plants had withered and died, and the pith became dark red (similar to field plants) (Figure 1). The fungi isolated from the experimental plants were confirmed as F. falciforme using morphological and sequence analyses. F. falciforme causes root rot in several species including Nicotiana tabacum (Qiu et al. 2023) and Weigela florida (Shen et al. 2020); however, this study is the first to report root rot caused by F. falciforme in strawberries in China. Overall, F. falciforme infection poses a threat to strawberry production and breeding.

Influence of flavonoids from Sedum aizoon L. on mitochondrial function of Rhizopus nigricans in strawberry.

Rhizopus nigricans (R. nigricans), one of the fungi that grows the fastest, is frequently discovered in postharvest fruits, it's the main pathogen of strawberry root rot. Flavonoids in Sedum aizoon L. (FSAL) is a kind of green and safe natural substance extracted from Sedum aizoon L. which has antifungal activity. In this study, the minimum inhibitory concentration (MIC) of FSAL on R. nigricans and cell apoptosis tests were studied to explore the inhibitory effect of FSAL on R. nigricans. The effects of FSAL on mitochondria of R. nigricans were investigated through the changes of mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), Ca2+ content, H2O2 content, cytochrome c (Cyt c) content, the related enzyme activity and related genes of mitochondria. The results showed that the MIC of FSAL on R. nigricans was 1.800mg/mL, with the addition of FSAL (1.800mg/mL), the mPTP openness of R. nigricans increased and the MMP reduced. Resulting in an increase in Ca2+ content, accumulation of H2O2 content and decrease of Cyt c content, the activity of related enzymes was inhibited and related genes were up-regulated (VDAC1, ANT) or down-regulated (SDHA, NOX2). This suggests that FSAL may achieve the inhibitory effect of fungi by damaging mitochondria, thereby realizing the postharvest freshness preservation of strawberries. This lays the foundation for the development of a new plant-derived antimicrobial agent.

Biocontrol Potential of Trichoderma asperellum CMT10 against Strawberry Root Rot Disease

Strawberry root rot caused by Neopestalotiopsis clavispora is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Currently, the only accessible control methods are fungicide sprays, which could have an adverse effect on the consumers of the strawberries. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides. Trichoderma spp. are frequently used as biological agents to prevent root rot in strawberries. In order to provide highly effective biocontrol resources for controlling strawberry root rot caused by Neopestalotiopsis clavispora, the biocontrol mechanism, the control effects of T. asperellum CMT10 against strawberry root rot, and the growth-promoting effects on strawberry seedlings were investigated using plate culture, microscopy observation, and root drenching methods. The results showed that CMT10 had obvious competitive, antimycotic, and hyperparasitic effects on N. clavispora CMGF3. The CMT10 could quickly occupy nutritional space, and the inhibition rate of CMT10 against CMGF3 was 65.49% 7 d after co-culture. The inhibition rates of volatile metabolites and fermentation metabolites produced by CMT10 were 79.67% and 69.84% against CMGF3, respectively. The mycelium of CMT10 can act as a hyperparasite by contacting, winding, and penetrating the hyphae of CMGF3. Pot experiment showed that the biocontrol efficiency of CMT10 on strawberry root rot caused by Neopestalotiopsis clavispora was 63.09%. CMT10 promoted strawberry growth, plant height, root length, total fresh weight, root fresh weight, stem fresh weight, and root dry weight by 20.09%, 22.39%, 87.11%, 101.58%, 79.82%, and 72.33%, respectively. Overall, this study showed the ability of T. asperellum CMT10 to control strawberry root rot and its potential to be developed as a novel biocontrol agent to replace chemical fungicides for eco-friendly and sustainable agriculture.

Complete genome sequence of a novel partitivirus with a dsRNA3 segment, isolated from Fusarium commune strain CP-SX-3 causing strawberry root rot.

A novel partitivirus, Fusarium commune partitivirus 1 (FcoPV1), was identified in Fusarium commune strain CP-SX-3 isolated from diseased roots of strawberry with symptoms of root rot. The complete genome of FcoPV1 comprises three double-stranded RNAs (dsRNAs): dsRNA1 (1,825 nt), dsRNA2 (1,592 nt), and dsRNA3 (1,421 nt). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), and dsRNA2 contains a single ORF (ORF2) encoding a coat protein (CP). dsRNA3 is a possible satellite RNA that does not appear to encode a known protein. BLASTp analysis revealed that RdRp (86.59%) and CP (74.13%) encoded by the two ORFs (ORF1 and ORF2) had the highest sequence similarity to their counterparts in Fusarium equiseti partitivirus 1 (FePV1). Phylogenetic analysis based on the complete amino acid sequence of RdRp suggested that FcoPV1 should be considered a member of a new species in the proposed genus "Zetapartitivirus" within the family Partitiviridae. To the best of our knowledge, this is the first report of a zetapartitivirus infecting phytopathogenic F. commune.

Binucleate Rhizoctonia AG‐A causing black root rot of strawberry in Albania

AbstractStrawberry production in Albania is very important and growing rapidly, while data on strawberry diseases are lacking. In 2022, symptoms of leaf necrosis, root rot, and wilt were observed on strawberry plants of cultivars ‘Savana’, ‘Sabrina’, and ‘Fortuna’ in greenhouses near Vajkan, Fier, Albania. The disease incidence was estimated at 50%. Only isolates of Rhizoctonia spp. were obtained from symptomatic plants. The isolates formed fast‐growing, white colonies with binucleate, typically branching hyphae and no sclerotia, resembling Rhizoctonia AG‐A. BLAST analyses of the ITS rDNA of isolate RhizAl (accession number OR231136) revealed the highest nt homology of 99%–100% (100% query coverage) with binucleate Rhizoctonia AG‐A isolates, while the neighbour‐joining phylogenetic analyses confirmed the identification. The pathogenicity of the isolate RhizAl was demonstrated by inoculations of disease‐free strawberry daughter plants (cv. ‘Fortuna’) that developed mild wilting and leaf necrosis 45 days after inoculation while plants collapsed with root necrosis 60 days after inoculation. In this study, binucleate Rhizoctonia AG‐A was detected for the first time as a causal agent of black root rot of strawberries in Albania and in general, and studies on the general importance of Rhizoctonia spp. in different crops in Albania are needed.

Preventive and Curative Effects of Treatments to Manage Strawberry Root and Crown Rot Caused by Neopestalotiopsis rosae.

Strawberry root and crown rot caused by the fungus Neopestalotiopsis rosae is an emerging disease that has caused yield losses of up to 70% in Mexico and other regions worldwide. This research evaluated the effects of biological and chemical fungicides applied as preventive and curative applications for controlling root and crown rot caused by N. rosae in strawberries under greenhouse conditions. Treatments included these chemical fungicides: prochloraz, prochloraz + thiram, cyprodinil + fludioxonil, difenoconazole + azoxystrobin, iprodione, captan, thiram, pydiflumetofen + fludioxonil, fluxapyroxad + pyraclostrobin, and hymexazol; each was applied at commercial doses. Also tested were biological treatments based on Trichoderma koningiopsis, T. asperellum, Streptomyces sp., and Bacillus amyloliquefaciens strain D747 (B. velezensis). Disease incidence, severity, plant mortality, root length, and dry weight were determined. Results showed that overall, preventive applications of the fungicides pydiflumetofen + fludioxonil, cyprodinil + fludioxonil, and prochloraz resulted in the smallest area under the disease progress curve and the lowest final disease incidence, severity, and plant mortality. An intermediate group of effective treatments entailed hymexazol, iprodione, T. asperellum, and T. koningiopsis (50 to 75% efficacy). Treatments with greater efficacy (99 to 100%), pydiflumetofen + fludioxonil, cyprodinil + fludioxonil, and prochloraz, also had maximal total plant biomass vis-à-vis the untreated control. By contrast, each treatment's efficacy was significantly reduced when applied curatively (0 to 37% treatment efficacy). These results suggest that certain treatments are useful for controlling strawberry root and crown rot caused by N. rosae when applied preventively (as root dipping). These results will contribute to designing more effective management programs for root and crown rot caused by N. rosae on strawberry.

Rhizosphere microbial ecological characteristics of strawberry root rot.

Strawberry (Fragaria × ananassa Duch.) holds a preeminent position among small fruits globally due to its delectable fruits and significant economic value. However, strawberry cultivation is hampered by various plant diseases, hindering the sustainable development of the strawberry industry. The occurrence of plant diseases is closely linked to imbalance in rhizosphere microbial community structure. In the present study, a systematic analysis of the differences and correlations among non-culturable microorganisms, cultivable microbial communities, and soil nutrients in rhizosphere soil, root surface soil, and non-rhizosphere soil of healthy and diseased strawberry plants affected by root rot was conducted. The goal was to explore the relationship between strawberry root rot occurrence and rhizosphere microbial community structure. According to the results, strawberry root rot altered microbial community diversity, influenced fungal community composition in strawberry roots, reduced microbial interaction network stability, and enriched more endophytic-phytopathogenic bacteria and saprophytic bacteria. In addition, the number of bacteria isolated from the root surface soil of diseased plants was significantly higher than that of healthy plants. In summary, the diseased strawberry plants changed microbial community diversity, fungal species composition, and enriched functional microorganisms significantly, in addition to reshaping the microbial co-occurrence network. The results provide a theoretical basis for revealing the microecological mechanism of strawberry root rot and the ecological prevention and control of strawberry root rot from a microbial ecology perspective.

First Report of Strawberry Black Root Rot Caused by Globisporangium sylvaticum in Tennessee, USA.

Globisporangium sylvaticum (syn. Pythium sylvaticum), is an oomycete that causes root rot and damping off of field crops, ornamentals, and vegetables. Several species in Pythiaceae are associated with black root rot of strawberry [(Fragaria × ananassa) Duchesne] (Millner 2006). Mature, stunted 'Chandler' strawberry plants, with reduced shoot vigor and black necrotic roots, were collected from Rhea County (June 2018) and Cumberland County, TN (May 2019). Aboveground symptoms occurred in low incidence (<5% of plants) in the fields. Plant roots were rinsed with tap water, cut into 1 to 3 cm pieces, and surface-disinfested (70% ethanol, 1 min) followed by a sterile water rinse. Root segments were crushed, placed on 20% V8 juice agar, and incubated in the dark at 21°C for 3 days. White fluffy mycelia grew from a majority of roots and coenocytic hyphae with globose hyphal swellings, delimited from hyphae by septa, were observed with microscopy. Hyphae were initially branched, curled, hyaline, and aseptate; however, septations were observed in older cultures. Globose structures (terminal and intercalary) were identified as sporangia [11 to 32 (avg. 22.1) µm diameter] when zoospores were observed (Parikh et al. 2022). Oospores [9 to 21 (avg. 16) μm diameter] were globose, smooth, aplerotic, and thick-walled. Oogonia, with or without one or more inflated antheridia, were observed when isolates were paired in culture, characteristics consistent with descriptions of Campbell and Hendrix (1967), Pratt and Green (1971), van der Plaats-Niterink (1981), and Uzuhashi et al. (2010). Genomic DNA was extracted (Extract-N-Amp™; Sigma-Aldrich, MO) for PCR amplification of internal transcribed spacer (ITS) regions of rDNA with primers ITS1/ITS4 (White et al. 1990); ITS and large subunit rRNA regions with primers UN-up18S42/UN-lo28S22 (Robideau et al. 2011); and cytochrome c oxidase subunit I (COI) mitochondrial DNA with primers OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011). Primers ITS1/ITS4 were used to amplify isolate TN (GenBank Accession MW386310, which had 100% homology with reference isolate MK326528). Primers UN-up18S42/UN-lo28S22 amplified isolates SAP18 and OO1 (Accessions MZ881935 and MZ881936, which had 99.8% homology with HQ665236), and COI primers amplified isolate SAP18 (Accession OK020192, which had 100% homology with GU071816 and KT692835). To satisfy Koch's postulates, inoculum of G. sylvaticum grown on autoclaved wheat seeds was added (5% w/v) to planting mix (1 peat:1 sand, v/v). Young, rooted strawberry plants were planted in 1.2-L pots with infested (n = 6) and control (no pathogen, n = 6) mixes, which was saturated with deionized water. Pots were covered with clear plastic for 48 h to maintain high humidity. Plants were grown in a greenhouse (24°C avg.) for 8 weeks. The disease assay was repeated. All plants in infested mix died, with black, necrotic roots. Plants in the control mix were healthy and well-established. The pathogen was reisolated from roots of all inoculated plants and confirmed to be G. sylvaticum based on morphology and molecular analyses. Root disease of strawberry caused by G. sylvaticum has been reported in the USA (Campbell and Hendrix 1967; Nemec and Sanders 1970; Pratt and Green 1971). This is the first report of G. sylvaticum causing root rot of strawberry in Tennessee. With the loss of methyl bromide, sustainable disease control strategies are needed to provide effective management options for strawberry black root rot.

First report of Fusarium keratoplasticum causing strawberry root rot in Sinaloa, Mexico.

Strawberry (Fragaria × ananassa) is a fruit of economic importance for Mexico, occupying the third place in world production, with an approximate production of 861, 337 t (SIAP, 2021). In January 2021, in Culiacan, Sinaloa, Mexico (24°46'46″N; 107°27'04″ W), wilting symptoms (stunted growth, leaf yellowing and wilting, necrosis in vascular bundles, root rot and wilting) were observed on commercial strawberry crops, with an incidence of 5 to 10 %. Tissue samples from symptomatic roots were cut and disinfected with alcohol, sodium hypochlorite and sterile water, to later be plated on potato dextrose agar (PDA). Fifteen monosporic isolates were obtained by single-spore culturing (Hansen and Smith, 1932). Typical Fusarium spp. colonies were obtained from all root samples. On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septate, and light-yellow pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were slightly curved, showing three marked septa, broad central cells, slightly tapered apices, foot-shaped basal cells and measured 59.6 - 73.4 (x̄ = 68.7) x 10.4 - 14.9 μm (x̄ = 13.6) (n = 40). The microconidia (n = 40) were thin-walled, hyaline, ovoid unicellular that measured 19.7 - 32.2 (x̄ = 26.6) x 8.8 - 11.8 μm (x̄ = 10.2). The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequence from the isolate FKTFRESCULSIN (GenBank accession no. OK491929) and other Neocosmospora and Fusarium species. Phylogenetic analysis revealed the isolate was Fusarium keratoplasticum (currently named Neocosmospora keratoplastica) belonging to the Fusarium solani species complex (FSSC). Pathogenicity tests were performed on strawberry plants (cultivar Camarosa) grown on autoclaved sandy loam soil mix. Twenty plants were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) in an isotonic saline solution of FKTFRESCULSIN grown on PDA. Ten uninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the field was observed. No symptoms were observed on uninoculated control plants after 60 days. The pathogen was reisolated from necrotic tissue from all inoculated plants and identified as F. keratoplasticum by sequencing the partial EF1-α gene and based on its morphological characteristics, thus fulfilling Koch's postulates. To our knowledge, this is the first report of root rot and wilt of strawberry caused by F. keratoplasticum in Mexico; it also contributes knowledge to the scientific community, since there is little information about this pathogen causing damage to plants in the world. Strawberry is an important crop in Mexico, and the occurrence of this disease could threaten strawberry production.

First Report of Neopestalotiopsis rosae Causing Crown and Root Rot of Strawberry in California.

Strawberry production in California represents over 38,000 acres with an annual farm value of $1.99 billion. Strawberry dieback was observed in February of 2021 in the Salinas Valley in central California. Disease symptoms included dead and dying 'Maverick' strawberry plants with necrotic lesions and black discoloration of the crown, root cortex, epidermis, and vascular tissues. Disease incidence was estimated to be 60% of a 20-acre field. The causal agent was isolated from five randomly selected symptomatic plants by surface disinfesting symptomatic crowns and roots in 1% sodium hypochlorite for 30 s, rinsed twice in sterile water for 30 s then placed on 3.7% potato dextrose agar (PDA) Petri dishes amended with 100 mg/L streptomycin, and then incubated for 7 days at 24°C under a 12-h photoperiod. Consistent white cottony fungal colonies were hyphal tip transferred to fresh PDA dishes and incubated as above for morphological and genetic comparisons. Black acervuli developed 7 to 9 days after incubation. Conidia were ellipsoidal, measuring 25 to 30 × 7.5 to 10 µm (average 26.8 × 9.2 µm, n = 30), with five cells. Apical and basal cells were hyaline, and the three median cells were versicolorous brown, with a single, straight, centric basal appendage and 3 to 4 flexuous apical appendages. Colony diameter averaged 90 mm in 7 days. Based on colony and conidial characters, the fungus was tentatively identified as a species of Neopestalotiopsis (Maharachchikumbura et al. 2014). Total genomic DNA was extracted from three axenic cultures using the Invitrogen Easy-DNA kit. Three genetic loci were PCR amplified and sequenced: internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factor 1-alpha (TEF) utilizing the primer pairs ITS1/ITS4, T1/Bt2a, and EF1-688F/EF1-1251R, respectively (White et al. 1990, O'Donnell and Cigelnik 1997, Glass and Donaldson 1995, and Alves et al. 2008). A BLASTn search of NCBI showed 99.6% identity (495/497 bases; OM942910-OM942912) with the type specimen of Neopestalotiopsis rosae CBS 101057 for the ITS locus. Both BT (765/765 bases, OM964802-OM964804) and TEF (475/475 bases; OM964799-OM964801) sequences were 100% identical to CBS 101057. Conidia of isolate PAR027 were scraped from the surface of 14-day-old PDA Petri dishes and inoculated (1 × 106 spores/mL: 2 mL/plant) to four apparently healthy strawberry transplant roots of the cultivar 'Monterey' in 'sunshine mix' potting soil. Two control plants were inoculated with sterile water. The experiment was conducted twice. Strawberry plants were maintained in a hoop house for four weeks, after which dieback and wilt symptoms resembled the symptoms observed in the field. Control plants remained asymptomatic and no pathogens were isolated. Fungal recovery from inoculated plants morphologically matched the original inoculum; thus, Koch's postulates was satisfied. To our knowledge, this is the first report of N. rosae causing crown and root rot disease of strawberry in California. Previously, N. rosae has been reported to cause serious decline of strawberry plants in Florida and several countries (Baggio et al. 2021, Rebollar-Alviter et al. 2020, Wu et al. 2021, Sun et al. 2021). Correct identification of the causal agent provides a proper foundation to identify control strategies for this emerging disease, which has the potential to become a significant problem for strawberry growers in the Salinas Valley of California.

Genetic Diversity and Population Structure of Fusarium commune Causing Strawberry Root Rot in Southcentral China

Strawberry plants and fruits are vulnerable to infections by a broad range of pathogens and pests. However, knowledge about the epidemiology of pathogens causing strawberry diseases is limited. In this study, we analyzed Fusarium commune, a major fungal pathogen causing strawberry root rot, from diseased strawberry root tissues in southcentral China. A total of 354 isolates were obtained from 11 locations that spanned about 700 km from both south to north and east to west. Multilocus genotypes of all isolates were obtained using seven polymorphic simple sequence repeat markers developed in this study. Our analyses revealed significant genetic diversity within each of the 11 local populations of F. commune. STRUCTURE analysis revealed that the optimal number of genetic populations for the 354 strains was two, with most local geographic populations containing isolates in both genetic clusters. Interestingly, many isolates showed allelic ancestry to both genetic clusters, consistent with recent hybridization between the two genetic clusters. In addition, though alleles and genotypes were frequently shared among local populations, statistically significant genetic differentiations were found among the local populations. However, the observed F. commune population genetic distances were not correlated with geographic distances. Together, our analyses suggest that populations of F. commune causing strawberry root rot are likely endemic to southcentral China, with each local population containing shared and unique genetic elements. Though the observed gene flow among geographic regions was relatively low, human activities will likely accelerate pathogen dispersals, resulting in the generation of new genotypes through mating and recombination.

PCR diagnostics for rapid detection of fungi associated with black root rot of strawberries

Black root rot is a disease of the strawberry with increasing importance for Germany. Until the early years of the twenty-first century, very different pathogens, including fungi and nematodes, were thought to cause this disease. However, based on substantial research between 2007 and 2014 at the fruit-growing center Jork (Esteburg, Germany) fungi with Cylindrocarpon-like anamorphs, i.e., Dactylonectria torresensis and Ilyonectria spp. were identified as causal agents. Both fungi are soil-borne pathogens and able to infect young strawberry plants in nurseries. Therefore, it is crucial to test plants prior to further propagation for the presence of these organisms. Although speed of diagnosis is a well-known and important factor, until today only time-consuming microbiological tests are available. To improve the situation, we aimed at developing a rapid, PCR-based, assay for the detection of fungi associated with black root rot. Most challenging was the design of specific primers that recognize the pathogen(s) in question while being unspecific for other fungi and Oomycetes routinely found on strawberries. Here, we report on a pair of primers that fulfill these rigorous criteria and present data on their specificity and sensitivity. A concentration of as low as 1.25 pg µl−1 template DNA from target pathogens could be detected in infected tissue. Significantly, the same primers were successfully used in standard and quantitative PCR assays. Based on our results, diagnostic laboratories are now enabled to rapidly survey lots of strawberry plants and provide growers with precise information on the presence of fungi associated with black root rot even in asymptomatic plants.

Morphological and molecular characteristics of fungal species associated with crown rot of strawberry in South Korea.

Crown and root rot is the most important and destructive strawberry diseases in Korea as it causes substantial economic loss. In August 2020, a severe outbreak of crown and root rot on strawberries (Fragaria × ananassa Duch.) was observed in the greenhouse at Sangju, South Korea. Infected plantlets displayed browning rot within the crown and root, stunted growth, and poor rooting. Thirty fungal isolates were obtained from the affected plantlet. Isolates were identified based on morphological characteristics and pathogenicity test as well as sequence data obtained from internal transcribed spacer, large subunit ribosomal ribonucleic acid, translation elongation factor, and RNA polymerase II-second largest subunit. Results showed that the crown and root rot of strawberry in Korea was caused by three distinct fungal species: Fusarium oxysporum f. sp. fragariae, F. solani, and Plectosphaerella cucumerina. To the best of our knowledge, F. solani, and P. cucumerina are reported for the first time as the causal agents of the crown and root rot of strawberry in South Korea. Pathogenicity tests confirmed that these isolates are pathogenic to strawberry. Understanding the composition and biology of the pathogen population will be helpful to provide effective control strategies for the disease.

Dactylonectria species associated with black root rot of strawberry in China

Dactylonectria species associated with black root rot of strawberry in China

Molecular reassessment of diaporthalean fungi associated with strawberry, including the leaf blight fungus, Paraphomopsis obscurans gen. et comb. nov. (Melanconiellaceae)

ABSTRACTPhytopathogenic fungi in the order Diaporthales (Sordariomycetes) cause diseases on numerous economically important crops worldwide. In this study, we reassessed the diaporthalean species associated with prominent diseases of strawberry, namely leaf blight, leaf blotch, root rot and petiole blight, based on molecular data and morphological characters using fresh and herbarium collections. Combined analyses of four nuclear loci, 28S ribosomal DNA/large subunit rDNA (LSU), ribosomal internal transcribed spacers 1 and 2 with 5.8S ribosomal DNA (ITS), partial sequences of second largest subunit of RNA polymerase II (RPB2) and translation elongation factor 1-α (TEF1), were used to reconstruct a phylogeny for these pathogens. Results confirmed that the leaf blight pathogen formerly known as Phomopsis obscurans belongs in the family Melanconiellaceae and not with Diaporthe (syn. Phomopsis) or any other known genus in the order. A new genus Paraphomopsis is introduced herein with a new combination, Paraphomopsis obscurans, to accommodate the leaf blight fungus. Gnomoniopsis fragariae comb. nov. (Gnomoniaceae), is introduced to accommodate Gnomoniopsis fructicola, the cause of leaf blotch of strawberry. Both of the fungi causing leaf blight and leaf blotch were epitypified. Fresh collections and new molecular data were incorporated for Paragnomonia fragariae (Sydowiellaceae), which causes petiole blight and root rot of strawberry and is distinct from the above taxa. An updated multilocus phylogeny for the Diaporthales is provided with representatives of currently known families.