Infected Strawberry Plants Research Articles

Native bacteria for field biocontrol of black root rot in Egyptian strawberry

BackgroundIncreasing cultivation of strawberry in Egypt has boosted efforts to increase its yield. Biocontrol agents (BCAs) may avoid side effects and health risks caused by chemical fungicides used to control black root rot disease in strawberry. Some BCAs control the disease and augment strawberry yield, but additional research is needed to fit BCAs into emerging control strategies. The impact of six bacterial isolates of Paenibacillus polymyxa and Bacillus brevis on this disease and on berry yield is reported and compared to a common chemical fungicide.ResultsThe bacterial isolates reduced the growth of the black root rot causal agents Fusarium solani, Rhizoctonia solani, and Macrophomina phaseolina, in dual culture bioassays. The greatest fungal inhibition was caused by P. polymyxa isolates 1 and 2 and B. brevis isolate 2. They suppressed the growth of F. solani, R. solani, and M. phaseolina by more than 74, 76, and 79%, respectively. Disease severity and incidence were significantly reduced on naturally infected strawberry plants in the field by the six isolates. The best results were obtained by the superior bioassay isolates which suppressed the disease incidence by 73, 77, and 71%, and its severity by 72, 78, and 70%, respectively. Disease suppression by bacteria was comparable to that by fungicide Actamyl. Bacteria surpassed Actamyl with regard to strawberry yield and enhancement of peroxidase and chitinase activities in the leaves.ConclusionsThese isolates are potential benign alternatives to fungicides used against black root rot in strawberry in Egypt. More studies are needed to examine their economic use on a wider scale.

Using Some Natural Substrates to Enhance the Growth, Yield and the Tolerance of Strawberry Plants to Rhizoctonia Root Rot Disease

Experiments of two seasons (2019/2020 and 2020/2021) were carried out in greenhouse at Department of Plant Pathology, Faculty of Agriculture, Ain Shams University. While, field experiments were carried out at El-Kanater Horticulture Research Station (El-Kaluobia Governorate), Horticulture Research Institute, Agriculture Research Center (ARC), Egypt.Compared with untreated infected strawberry plants Festival cultivar, all applications with chitosan at 500 ppm, methionine at 20 ppm and ascorbic acid at 20 mM via seedling root soaking, foliar spray, and combining the root soaking with foliar spray, or seedling root soaking with Rhizolex (1g/L) showed significant decrease in root rot incidence caused by Rhizoctonia solani. The combining of root soaking with foliar spray of chitosan or methionine resulted the highest significant decreases of disease incidence under greenhouse and field conditions. However, all applications except Rhizolex resulted in significant increases in phenolic compounds, oxidative enzymes activity i.e., PPO, SOD and CAT in roots during the two growing seasons. Obtained results showed that soaking plus foliar spraying with chitosan at 500ppm, methionine at 20 ppm and ascorbic acid at 20 mM significantly increased all vegetative growth characters, N, P, K and total carbohydrate in plant foliage, fruit yield and its components as well as physical and chemical fruit quality.

Genetic Diversity of Seven Strawberry mottle virus Isolates in Poland.

The studies on detection of the Strawberry mottle virus (SMoV) have been conducted in Poland for breeding programme purpose and for producers of strawberry plant material. Leaf samples collected from infected strawberry plants were grafted on Fragaria sp. Indicators which were maintained in greenhouse for further study. Seven Fragaria vesca var. semperflorens ‘Alpine’ indicators infected by SMoV were used for the study aimed on molecular characterization of virus isolates. Partial RNA2 was amplified from total nucleic acids using the RT-PCR method. The obtained amplicons separately digested with BfaI, FauI, HaeIII, HincI, and TaqI enzymes showed different restriction profiles. The nucleotide sequences analysis of RNA2 fragment confirmed the genetic diversity of the SMoV isolates as their similarity ranged from 94.7 to 100%. Polish isolates shared 75.7–99.2% identity with sequence of the virus strains from the Czech Republic, the Netherlands, and Canada. Phylogenetic analysis resulted in grouping of the isolates found in Poland together with one of the Czech strain whereas two other from the Czech and the strains from the Netherlands and Canada created the separate cluster.

Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil.

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

BackgroundStrawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas.FindingsThe complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria × ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86–100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20–40 % in 8–9 weeks post-inoculation using syringe-inoculation.ConclusionsThe complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0624-1) contains supplementary material, which is available to authorized users.

Rapid detection of Colletotrichum gloeosporioides in infected strawberry plants using loop-mediated isothermal amplification

We developed a loop-mediated isothermal amplification (LAMP) assay for detecting Colletotrichum gloeosporioides, which causes anthracnose, in strawberry plants. Primer sets targeted the region from the gene encoding 5.8S ribosomal RNA to internal transcribed spacer 2, which is highly conserved in 25 Colletotrichum species in GenBank. Our results indicated that the LAMP assay, which uses template DNA prepared using microwave irradiation, is a simple and sensitive tool for detecting C. gloeosporioides in infected strawberry plants. Further, our results showed that the LAMP assay could differentiate Colletotrichum species such as C. acutatum, C. aenigma, and C. siamense.

Response of some Commercial Strawberry Cultivars to Infection by Wilt Diseases in Egypt and their Control with Fungicides

Isolation trials carried out from naturally infected strawberry plants by wilt diseases grown in three Counties at Qalubiya Governorate resulted in the presence of four fungi with different frequencies. These fungi were identified as Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani and Pythium sp. Pathogenicity test using the four fungi showed that only F. oxysporum and V. dahliae were able to cause the typical symptoms of wilt. Evaluation of six strawberrycultivars against F. oxysporum and V. dahliae under greenhouse conditions revealed that cvs. Festival, Florida and Sweet Charlie were less susceptible. While under field conditions they exhibited low percentages of final disease severity and low values of area under disease progress curve (AUDPC), low values of relative area under disease progress curve (rAUDPC) and low rates of infection (r-values) during the two tested growing seasons of 2010/11 and 2011/12. On the other hand, cvs. Fortuna and Camarosa exhibited the opposite results in this respect. Six fungicides were evaluated on the susceptiblecv. Camarosa under greenhouse conditions and for two successive seasons under field conditions (2011/12 and 2012/13) where the most effective fungicides were Uniform, Tachigaren and Topsin-M in controlling wilt infection. The highest yield increase percentage was recorded in cv. Camarosa during the two growing seasons (2012/13 and 2013/14) under protective trial by Uniform fungicide.

Survival, Host-Pathogen Interaction, and Management of Macrophomina phaseolina on Strawberry in Israel.

Crown and root rot of strawberry, caused by Macrophomina phaseolina, have become predominant soilborne diseases of strawberry in Israel over the past 5 years. In total, 151 isolates of the pathogen were isolated from infected strawberry plants of commercially grown cultivars in Israel onto a modified agar medium for the genus Macrophomina. Sclerotia viability declined more rapidly in soil maintained at 25°C or at soil temperatures fluctuating from 18 to 32°C under greenhouse conditions, compared with sclerotia viability in soil kept at 30°C. After 30 to 40 weeks of exposure in soil, inocula maintained at 25 or 30°C or at fluctuating temperatures in a greenhouse declined to negligible levels. A significant increase in plant mortality was observed in infested soils maintained at 30 versus 25°C, whereas water stress at 25 or 30°C did not affect plant mortality in M. phaseolina-infested soils. This demonstrated the importance of elevated soil temperature, not moisture stress, on plant mortality caused by M. phaseolina. Host specificity was not evident when strawberry plants were inoculated with each of seven Israeli isolates of M. phaseolina obtained from six other plant species, suggesting the importance of keeping strawberry crops out of rotation with other host crops of the pathogen. The soil fumigants methyl bromide (applied at 500 kg/ha) and metam sodium (730 liter/ha) caused 90 and 95% pathogen mortality in field experiments, respectively, indicating that fumigation may be an effective method of managing this pathogen in infested soils. The increase in prevalence of crown and root rot caused by M. phaseolina in strawberry crops in Israel may be related to the phase-out of methyl bromide.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Strawberry in Sweden.

Strawberries (Fragaria × ananassa) are the most important soft fruit crop in Sweden and most commonly grown as a perennial crop. Colletotrichum acutatum J.H. Simmond was detected on strawberries in Sweden for the first time in the autumn of 2003. Anthracnose caused by C. acutatum has previously been found on rhododendron in Sweden (2). The infected strawberry plants, cv. Kimberly, were found in a field in the northeastern part of Skåne in southern Sweden. The first plant sample was collected during September 2003, and a second sample was collected during the end of June 2004. Symptoms were seen on stolons as elliptical, black, sunken lesions and irregular black leaf spots originating from the tip. No symptoms were seen on immature green fruits. However, the farmer had observed black spots on the fruits the previous year. Immature fruits were collected in the field, surface sterilized with 70% ethanol, dipped for 1 min in 1.25% NaOCl, and thereafter rinsed in distilled water. Fruits were incubated in a moist chamber at 23°C. The first symptoms were seen as black spots after 3 days; later, salmon-colored conidial masses were produced from acervuli in the center of the lesions. The fungus was isolated on potato dextrose agar (PDA) as single-conidia cultures. Colonies were white-to-pale gray with abundant salmon-colored masses of conidia in the center of the culture. Mycelial growth rate on PDA was 7.5 to 7.9 mm per day at 21 ± 1°C. Conidia (11.0 to 16.5 × 3.9 to 5.7 μm) were cylindrical and hyaline with pointed ends, which is consistent with previous reports for C. acutatum (1). Symptoms were reproduced by artificial inoculation on ripe strawberries (cv. Elsanta). A conidia suspension was prepared from single-conidia cultures and each strawberry was sprayed with 0.5 ml of suspension (2.2 × 107 conidia per ml). The strawberries were incubated in a moist chamber at 22°C. The first typical symptoms were seen after 3 days. Single conidia were isolated from infected fruits and plated on PDA. Stolons and leaves of cv. Alice were inoculated with mycelial plugs of C. acutatum, sealed in a plastic bag for 2 days, and then placed in a greenhouse environment at 70% relative humidity and 21°C. Black, sunken lesions were found on stolons after 7 days. A sample of 300 petioles from the infected field was sent to the Central Science Laboratory, York, England that identified the pathogen as C. acutatum using enzyme-linked immunosorbent assay and plating rinse water from petioles onto selective media. With this report, anthracnose caused by C. acutatum on strawberry has now been reported from all the Nordic countries.

DIAGNOSIS AND QUANTIFICATION OF STRAWBERRY VEIN BANDING VIRUS USING MOLECULAR APPROACHES

PCR and gel analyses were used for detection and quantification of Strawberry vein banding virus. A viral DNA isolation procedure was optimized to allow quick isolation of total nucleic acids prior to PCR. Different aged leaves, petioles, flowers and roots from infected strawberry plants including a virus indicator and three commonly grown cultivars were used to determine the most suitable tissues for virus testing and evaluate different diagnostic procedures. Nucleic acid preparations were diluted 10-10,000 fold in sterile water to determine the detection limits of PCR and the relative virus titer of samples based on dilution end point assay. The highest virus titer was in old symptomatic leaves and the lowest was in petioles. However, virus titer varied greatly among source plants used. To determine the PCR inhibition limits, concentrated preparations (up to 100x higher than those used in routine diagnosis) were used as templates. False negative results were interpreted as inhibition of Taq DNA polymerase activity by host derived contaminants with the highest levels in terminal roots and the lowest in young terminal leaves. These data were used to design a framework in mass screening field samples by pooling multiple samples. Colorimetric PCR and dot blot hybridization assay were used to confirm the above findings.

“Candidatus Phlomobacter fragariae” Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China)

ABSTRACT Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.

Detection of Strawberry crinkle virus in plants and aphids by RT‐PCR using conserved L gene sequences

About 10% of the large (L) protein gene of Strawberry crinkle virus (SCV) was sequenced after amplification with degenerate primers designed to conserved regions of the rhabdovirus L protein. The virus sequence was extended to 1362 nucleotides through rapid amplification of cDNA ends. One pair of degenerate L gene primers amplified a 683‐bp fragment from four different isolates of SCV cultured in the experimental host Physalis pubescens; the nucleotide sequences of these fragments differed by < 1% to 10% indicating the suitability of this region as a diagnostic target. This information enabled the development of a reverse transcription polymerase chain reaction (RT‐PCR) detection method for SCV using primers designed to the L gene sequence. SCV was amplified from infected P. pubescens (573 bp fragment) and from infected Chaetosiphon fragaefolii aphids (770 bp fragment). SCV was also detected by RT‐PCR in total RNA extracts from three strawberry plants showing symptoms typical of SCV infection but failed when the intensity of the disease symptoms decreased. However, both SCV positive‐sense RNA, and negative‐sense genomic RNA, were detected by nested PCR in chronically infected strawberry plants sampled in September.

Cloning of the spoT gene of "Candidatus Phlomobacter fragariae" and development of a PCR-restriction fragment length polymorphism assay for detection of the bacterium in insects.

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium "Candidatus Phlomobacter fragariae" is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between "P. fragariae" and other insect-associated proteobacteria, isolation of "P. fragariae" genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from "P. fragariae"-infected strawberry plants. It encodes part of a "P. fragariae" open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish "P. fragariae" from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry "P. fragariae." Interestingly however, the "P. fragariae" spoT sequence could be easily detected in whiteflies proliferating on "P. fragariae"-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.

Identification ofXanthomonas fragariaeField Isolates by rep-PCR Genomic Fingerprinting

Xanthomonas fragariae, the causal organism of angular leaf spot on cultivated strawberry (Fragaria x ananassa), is an economically important pathogen of nursery stock in California. The ability to reliably detect this pathogen in a timely manner is crucial for the production and timely distribution of disease-free nursery stock. Pathogenicity testing for this disease requires excessive time, and the bacterium grows slowly on standard culture medium. A medium, similar to that used for culturing Xylella fastidiosa, allowed more consistent recovery of X. fragariae from infected strawberry plants. Using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR), we generated genomic fingerprints of reference strains of X. fragariae (ATCC 33239 and 33240). These fingerprints were used, in turn, to accurately identify X. fragariae field isolates collected over the last 5 years from nurseries in California. The rep-PCR fingerprint results agree with pathogenicity test results, require much less time than the pathogenicity test, and have greater specificity than indirect enzyme-linked immunosorbent assay for identifying X. fragariae from field plants. For these reasons, rep-PCR is the fastest and most accurate method for the current identification of X. fragariae and it constitutes a useful tool for the production of disease-free strawberry nursery stocks.

Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10‐15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.

Estimation of the Distribution of Verticillium Populations in Infected Strawberry Plants and Soil

SUMMARYA rapid dilution‐plate technique has been evolved for the quantitative assay of the inoculum density of Verticillium spp. in infected plant tissues and in soil. Results showed a very high level of the pathogen (1,500 propagules/g soil) in the top 10 cm of soil from a severely‐infested strawberry site with the counts decreasing at lower depths. The top 30 cm of soil contained 98 per cent of the total estimated population.Abundant Verticillium inoculum was present in all vegetative tissues of infected plants with the exception of fruit stalks, the highest counts being recorded in root extracts. The relatively high inoculum level of the pathogen in stolon extracts demonstrates one method by which young runners may become infected.

Transmission of arabis mosaic virus by the nematode Xiphinema diversicaudatum (Micol.)

After dagger nematodes Xiphinema diversicaudatum (Micol.), were shown to transmit arabis mosaic virus to strawberry plants, the mode of transmission was studied using Petunia hybrida Vilm. seedlings as test plants. Approximately 30% of the nematodes reared on roots of infected strawberry plants transmitted the virus, and batches of ten nematodes caused infection in 90% of the test plants. Three days was the minimum test-feeding period in which transmission occurred, but longer periods resulted in a higher proportion of plants becoming infected. The vector retained the virus for long periods; some nematodes transmitted the virus to test plants in a second series without infecting those in the first on which presumably they fed for periods of 5, 10, and 20 days. Nematodes were still infective after fasting periods of up to 30 days in autoclaved sand. Noninfective nematodes acquired the virus in 24 hours, the minimum acquisition period tested, but a higher proportion were infective after 3 days on the source plants.