The relationship between colonisation and crown rot symptoms in strawberry plants infected with Phytophthora cactorum

Strawberry tissues infected with Phytophthora cactorum were comminuted and plated in a selective antibiotic agar medium to determine levels of tissue colonisation as indicated by the number of colony forming units (CFU) recovered per gramme of infected tissue. The number of CFU recovered per gramme of tissue increased logarithmically with the amount of necrosis in infected crown, leaf and petiole tissues. Under the conditions of enhanced susceptibility to infection and colonisation caused by cold storage treatments, this relationship between colonisation and necrosis was not significantly altered in the susceptible cv. Tamella. A recovery index was used to determine the effect of infected tissues on the recovery of CFU. This indicated that increasing levels of host colonisation stimulated CFU recovery and may partly explain the large increase in CFU g^-1 with larger amounts of necrosis. The amount of tissue colonisation was greater in inoculated plants of the susceptible cv. Tamella than in less susceptible cv. Cambridge Favourite, although the necrotic tissues of the latter contained more CFU g^-1, indicating a greater level of tolerance to colonisation. In cv. Tamella small amounts of colonisation were capable of causing wilt symptoms, although no wilted plants contained less than 200 CFU g^-1. Conversely, plants containing more than 1000 CFU g^-1 always wilted. In the early stages of infection, low levels of colonisation could be detected in strawberry crowns in the absence of symptoms. Dormant strawberry plants of cv. Tamella were readily infected by P. cactorum zoospore inoculations but, unlike actively growing plants, the majority of infections remained latent. These latent infections exhibited little or no symptoms and CFU recoveries from infected tissues were always below 100 CFU g^-1.     

Viral diversity in Phytophthora cactorum population infecting strawberry

Eighty-eight Phytophthora cactorum strains isolated from crown or leather rot of strawberry in 1971–2019 were screened for viruses using RNA-seq and RT-PCR. Remarkably, all but one isolate were virus-infected, most of them harbouring more than one virus of different genera or species. The most common virus occurring in 94% of the isolates was the Phytophthora cactorum RNA virus 1 (PcRV1) resembling members of Totiviridae. Novel viruses related to members of Endornaviridae, named Phytophthora cactorum alphaendornaviruses 1-3 (PcAEV1-3), were found in 57% of the isolates. Four isolates hosted viruses with affinities to Bunyaviridae, named Phytophthora cactorum bunyaviruses 1-3 (PcBV1-3), and a virus resembling members of the proposed genus ‘Ustivirus’, named Phytophthora cactorum usti-like virus (PcUV1), was found in a single isolate. Most of the virus species were represented by several distinct strains sharing ≥81.4% aa sequence identity. We found no evidence of spatial differentiation but some temporal changes in the P. cactorum virus community were observed. Some isolates harboured two or more closely related strains of the same virus (PcAEV1 or PcRV1) sharing 86.6%–96.4% nt identity in their polymerase sequence. This was surprising as viruses with such a high similarity are typically mutually exclusive.     

Double-stranded RNA analysis of strawberry plants affected by June yellows

Using an improved method for extraction of dsRNA from strawberry leaf tissue, small quantities of several dsRNA species with mol. wt greater than 1.0 × 10⁶were detected in strawberry plants free from known strawberry viruses but affected by June yellows (JY). No such dsRNA species were detected in plants of Fragaria vesca or seven strawberry cultivars known to be free from JY. Neither JY symptoms nor these dsRNA species were detected in healthy strawberry and F. vesca plants graft-inoculated with tissue from JY-affected plants. It is not known whether the JY-associated dsRNA species are those of a causal agent of JY or are a consequence of the JY condition. Nevertheless, the detection of such dsRNA species in plants affected by JY may offer a possible objective method for detecting the incipient condition in symptomless strawberry plants. However, the concentrations of dsRNA in JY-affected plants are very low and dsRNA analysis is thought not to be sufficiently reliable for routine testing of plants. The occurrence of anomalous dsRNA species in extracts from some strawberry plants was caused by dsRNA from two-spotted spider mites (Tetranychus urticae) infesting the plants.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Silicon influences growth and mycorrhizal responsiveness in strawberry plants

Effect of silicon (Si) on the response of strawberry (Fragaria?×?ananassa var. Parus) plants to arbuscular mycorrhizal fungus (AMF) was studied under growth chamber conditions. Plants were grown in perlite irrigated with nutrient solution without (??Si) or with (+?Si) 3 mmol L^?1 Si (~?84 mg L^?1 Si as Na₂SiO₃) in the absence (??AMF) or presence (+?AMF) of fungus. Dry matter production, root colonization rate, photosynthesis rate and water relation parameters were all improved by both Si and AMF, and the highest amounts were achieved by +?Si?+?AMF treatment. Mycorrhizal effectiveness increased by Si treatment associated with higher Si concentration in the +?AMF plants. Leaf concentrations of total soluble and cell wall-bound phenolics were increased by Si accompanied by the enhanced activity of phenylalanine ammonia lyase, but not polyphenol oxidase. Profile of phenolics compound revealed that gallic acid, caffeic acid, epicatechin, chlorogenic acid, ellagic acid and kaempferol increased by both Si and AMF treatments, while p-coumaric acid decreased. In addition to vegetative growth, both treatments improved fruit yield and its quality parameters. Our results showed that Si and AMF acted in a synergistic manner and improved growth and biochemical parameters in strawberry plants. However, the mechanism for Si-mediated increase of mycorrhizal effectiveness is not known, thereby needing further elucidation.     

Photocontrol of petiole elongation in light-grown strawberry plants

Summary The mode of phytochrome control of elongation growth was studied in fully-green strawberry (Fragaria x Ananassa Duch.) plants. Petiole growth showed two distinct types of response to light. In one, the end-of-day response, petioles were lengthened by low-intensity far-red irradiation for 1 h immediately following the 8 h photoperiod. The response was little or no greater with prolonged exposure and less when the start of far-red was delayed. It was already evident in the first leaf to emerge after treatment began. With the development of successive leaves a second, photoperiodic, type of response appeared, in which petioles lengthened following only prolonged exposure to red, far-red, mixtures of the two, or tungsten lighting, all at low levels of intensity. As with the inhibition of flowering in previous experiments, irradiation with red light during the second half of the otherwise long dark period gave the greatest response.Abbreviations and Symbols FR far-red light - HIR high irradiance response - R red light - P_r phytochrome in the red light absorbing form - P_fr phytochrome in the far-red light absorbing form - SDP short-day plant - LDP long-day plant - PAR photosynthetically active radiation     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

The production of strawberry plants from callus cultures

Shoots were regenerated from callus of the commercially important strawberry varieties Bogota, Brighton, Cambridge Favourite, Hapil, Ostara, Rapella, Red Gauntlet and JILA33 which is a promising selection from a current breeding programme.The callus was initiated from explants of petiole or lamina of leaves of micropropagated shoots in vitro or of lamina or peduncle from greenhouse plants. There was more shoot regeneration with callus from lamina than from petiole although with the variety Hapil, regeneration occurred only with callus from peduncle.With seven of the varieties, shoot regeneration occurred on culture media with BAP and 2,4-D whilst with the remaining variety, Cambridge Favourite, it occurred only with medium which contained 1AA- alanine conjugate in place of 2,4-D.Regenerated shoots rooted readily and the plants produced are being studied for somaclonal variation.     

Growth-promotion of strawberry plants inoculated with Azospirillum brasilense

Azospirillum brasilense (strains REC3, RLC1, PEC5) were root inoculated in strawberry plants of the cultivars ‘Milsei’, ‘Selva’ and ‘Camarosa’ to assess plant growth-promoting effects. The bacteria were able to promote plant growth (expressed as root length, root area, and dry weight of root and shoot), depending on the genotypes of plants and bacteria used, whereas the stolon production (3–4) depended only on the strawberry cultivar. To explain whether root exudates plays any role on the growth-promotion observed herein, total protein and sugar were determined, and chemotaxis properties were evaluated. The strains showed positive chemotaxis toward the root exudates, being influenced by the total sugars content, suggesting that the latter plays an important role in the chemotaxis effect and may contribute to enhance the root capacity to recruit azospirilla from rhizosphere, thus improving the growth-promoting effect exerted by these bacteria.     

Evaluation of methods of screening strawberry cultivars for resistance to crown rot caused by Phytophthora cactorum

Four methods were evaluated in measuring resistance of strawberry cultivars to crown rot caused by Phytophthora cactorum. Meristem propagated plants grown in vitro were inoculated with mycelial discs. Four to five days after inoculation, it was possible to distinguish between cultivars with large differences in susceptibility to the disease. Ten days later, all plants were totally necrotic making it impossible to distinguish between cultivars. When detached leaves were inoculated by inoculating a plug of mycelium into the petiole, disease symptoms developed more slowly in resistant cultivars, but leaf age greatly affected the rate of symptom development. When plug plants (not cold stored) were lightly wounded in the rhizome with a scalpel and inoculated with either zoospores or mycelium, differences in disease development between cultivars were mainly as would be expected from previous information on susceptibility, but both age and size of plants influenced the rate of disease development. Unwounded, inoculated plants did not develop symptoms. When cold‐stored plug plants were either unwounded or lightly wounded with a scalpel in the rhizome and inoculated with zoospores, the relative rates of disease development consistently reflected the susceptibility to crown rot. At the time of final assessment, disease was much more severe in wounded plants, but the relative susceptibility of cultivars was not affected by the wounding.     

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 g/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 g/ml kanamycin, but exhibited reduced rooting ability.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NPT-II neomycin phosphotransferase(EC 2.7.1.95) - GUS -glucuronidase(EC 3.2.1.31) - X-GLUC 5-bromo-4-chloro-3-indolyl-glucuronide - 4-MU 4-methylumbelliferone NRCC No. 31227     

Assessment of the antiproliferative activity on murine melanoma cells of extracts from elicited cell suspensions of strawberry,strawberry tree,blackberry and red raspberry

The antiproliferative activity of extracts from cell suspensions of strawberry (Fragaria x ananassa Duch.), blackberry (Rubus fruticosus L.), red raspberry (Rubus idaeus L.) and strawberry-tree (Arbutus unedo L.) on murine B16-F10 melanoma cells was investigated. To enhance the synthesis of the bioactive compounds, various elicitors, i.e. temperature (cold and cold/warm cycle), light (blue and red) and increased nitrogen concentration, were also applied to the cell cultures to evaluate the effects on their antiproliferative activity. The extracts from all the species reduced murine melanoma cell proliferation (between 30% and 38% relative to the control) and an improvement of antiproliferative activity on murine melanoma cells, relative to the un-elicited berry suspensions, was shown by the extracts from strawberry and strawberry-tree cells treated with red and blue light.     

Contribution of Gluconacetobacter diazotrophicus to phosphorus nutrition in strawberry plants

Plant and Soil - Gluconacetobacter diazotrophicus (family Acetobacteraceae), is a N2-fixing bacterium with capability of mineral solubilization through organic acid production. The aim of this work...