Somatic embryogenesis and shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium×P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes

Hairy roots were obtained after inoculation with Agrobacterium rhizogenes strain NCPPB 1855 of the in-vitro-grown shoots of the cherry rootstocks Colt (Prunus avium×P. pseudocerasus) and Mazzard F12/1 (P. avium L.). Not all putatively transgenic roots were able to grow in hormone-free medium. Mazzard F12/1 roots, induced with A. rhizogenes, did not differentiate any shoot or embryo, while both somatic embryos and shoots differentiated from the transgenic roots of Colt in medium containing 1 mg/l 6-benzylaminopurine and 1 mg/l 1-naphthaleneacetic acid. Somatic embryos were capable of secondary embryogenesis, but few developed into whole plants. DNA hybridization showed both a different number of bands and signal intensity in each of the five transgenic shoot clones and embryos examined. In a morphogenetic in vitro test, leaf explants of the transgenic shoot clones showed an increased capacity to differentiate roots, although clones differed in their sensitivity to the hormone ratio. Clones from the transgenic shoots had not only an increased rooting ability when grown in vitro but also exhibited various hairy root phenotypes when cultured in vitro and when transferred into the greenhouse. Received: 12 December 1996 / Revision received: 21 March 1997 / Accepted: 10 May 1997     

The role of Thielaviopsis basicola in the specific replant disorders of cherry and plum

The hypothesis that Thielaviopsis basicola might be the causal agent of the specific replant disorders of cherry and plum was tested in experiments with pot-grown trees and by observations on the distribution of the pathogen. The results obtained with certain strains of T. basicola simulated and were consistent with all features known to characterize the specific replant disorders, viz. inter- and intra-generic specificity of effect; host symptoms; establishment, immobility and persistence of the causal agent in soil; normal growth of affected trees after transfer to ‘non-replant’ soil; limited influence of soil type on the incidence of the disorder. T. basicola was therefore considered to be the probable causal agent of the specific replant diseases of certain Prunus species. The etiology of these diseases is discussed. T. basicola was neither pathogenic to apple nor associated with the growth of apple. The apparently closely similar specific replant disorder of Malus is therefore considered to be causally distinct from those of cherry and plum.     

Influence of Mycorrhizal Tobacco Roots, Incorporated Into the Soil, on the Development of Thielaviopsis basicola

The interaction between the soil-borne pathogen Thielaviopsis basicola and mycorrhizal roots infected by Glomus microcarpum has been studied during 4 months. A significant reduction in the number of propagules was observed in soils treated with mycorrhizal roots and an inhibition of T. basicola growth by a substance produced by mycorrhizal roots is suggested.     

Somatic hybridization of sexually incompatible top-fruit tree rootstocks,wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus)

Summary Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.     

Experimental Transmission of Plum Pox Virus (PPV) to Prunus Mahaleb and Prunus avium

Sharka caused by plum pox virus (PPV) is a disease spread in France since 1970, and causing severe damages essentially on apricot but also on plums and peach. Cherry is generally considered as not infected by PPV. Experimental transmissions by chip budding or aphids allowed to show that 3 isolates of PPV can multiply inside three cherry rootstocks (P. Mahaleb cv.‘SL 64′, P. avium cv.‘F 12-1′, and P. avium*P. pseudocerasus cv. ‘Colt') (Tables 1 and 2). But generally, the virus remained localized to the infection site and disappeared quickly (Table 3). Typical symptoms of chlorotic ringspot or vein clearing are also limited to the leaves probed by the aphids. The fact that no translocation was detected is discussed.     

Seed and soil: sources of inoculum for the colonisation of the foliage of solanaceous hosts by Pseudomonas solanacearum

Pseudomonas solanacearum was transmitted from contaminated seed to the cotyledons of capsicum (Capsicum frutescens) at 92, 73 and 60% r. h. and to the cotyledons of tomato (Lycopersicon esculentum) seedlings at 92% r. h. Subsequent epiphytic colonisation of the true leaves of capsicum occurred at 92 and 73% r. h. An increase in the population on capsicum cotyledons was detected at 92 and 73% r. h. but only at the higher r. h. was an increased population of P. solanacearum associated with the colonisation of the true leaves. Lesions developed on the true leaves of capsicum at 92% r. h. Transmission of P. solanacearum from capsicum seeds was affected by inoculum concentration, occurring at an infestation level of ∽ 10³ propagules seed^-1 but not at 50 propagules seed^-1. Pseudomonas solanacearum was detected on the cotyledons of capsicum held at 98% r. h. after germination of seed in soil infested with 2 × 10⁸ propagules g^-1 soil; lesions were detected on cotyledons 11 days after planting and invasion occurred in 10 stems and one root of the 20 seedlings sampled. The movement of the pathogen from the soil to the seedlings was affected by the level of soil infestation. Pseudomonas solanacearum was detected in four pairs of cotyledons, two stems and one root of the 36 seedlings sampled from soil infested with 10⁷ propagules g^-1 soil but it was not isolated from seedlings sampled from soil infested with 10⁵ or 4 × 10³ propagules g^-1 soil. Leaf and stem prints demonstrated the epiphytic nature of this organism on the cotyledons and stems of seedlings.     

持续干旱对樱桃根际土壤细菌数量及结构多样性影响

以1年生吉塞拉实生容器苗为试材,采用绿色荧光蛋白基因标记技术,研究了干旱胁迫(连续干旱0、7、14、21、28 d和35 d)对樱桃根际促生细菌YT3的标记菌YT3-gfp数量的影响,同时结合平板计数法和末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism,T-RFLP)技术,研究了干旱对樱桃土壤中的微生物数量及细菌群落结构多样性影响。结果表明:樱桃根际土壤中的YT3-gfp数量是非根际土壤中的8.75—28.77倍,随着持续干旱强度的增加,YT3-gfp的数量先增加后减小。干旱对根际土壤中YT3-gfp数量的影响大于对非根际土壤的影响,分别在持续干旱至第21天和28天时,YT3-gfp的数量达到最大值。随着持续干旱强度的增加,根际土壤中细菌和放线菌数量先增加后减小,而真菌的数量一直减少。此外,持续干旱至第21天或28天时,樱桃根际土壤具有最高的丰富度指数、多样性指数和最低的优势度指数。基于T-RFLP的主成分分析结果显示持续干旱14—35 d时,其细菌群落结构成为一个相对独立的群,群落结构趋于多样性;而持续干旱7 d和42 d构成另外两个相对独立的群,群落结构趋于简单。以上分析可知,干旱对土壤微生物影响显著,一定强度的干旱可提高细菌和放线菌数量,提高细菌群落结构多样性,适当干旱对维持根际土壤细菌群落结构多样性是有益的。     

Isozyme diversity in sour,sweet, and ground cherry

Thirty-six sour (Prunus cerasus L.), sweet (P. avium L.), and ground cherry (P. fruticosa Pall.) selections were evaluated for seven enzyme systems and principal coordinate analysis was used to examine isozyme divergence among these cherry species. The enzyme systems studied were phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD), leucine aminopeptidase (LAP), shikimate dehydrogenase (SKDH), and malate dehydrogenase (MDH). The first principal coordinate, which accounted for 41% of the total variation, separated the diploid sweet cherry selections from the sour, ground, and sour x ground cherry tetraploids. An additional 86 selections were evaluated for up to six of the enzyme systems to determine the polymorphisms at the enzyme loci and the level of heterozygosity between the diploid sweet cherry and the tetraploid species and interspecific hybrids. 6-PGD was the most polymorphic enzyme exhibiting 16 patterns. The tetraploid cherry species were more heterozygous than the diploid sweet cherry with an average heterozygosity of 78% compared to 19% for the diploids.     

Attraction, acquisition, retention and spatiotemporal distribution of soilborne plant pathogenic fungi by shore flies

Adult shore flies were experimentally shown to be aerial vectors for three soilborne plant pathogens: Verticillium dahliae, Fusarium oxysporum f.sp. basilici and Thielaviopsis basicola. Adult insects are attracted to sporulating cultures of the soilborne fungi investigated, as well as infected plant tissues. Shore flies acquired fungal propagules both by ingestion and surface contamination. The minimum acquisition time for propagules of soilborne fungi was 10–20 min and acquisition increased with time to reach 100% frass deposits infestation after 2 h of acquisition. The inoculum potential of the frass deposits was high considering that the number of viable spores deposited by one adult insect in a day was 2.38 × 10⁷, 3.08 × 10⁶ and 8.83 × 10⁶ for V. dahliae, F. oxysporum f. sp. basilici and T. basicola, respectively. Electron microscopy investigation implicated body surface contamination as a means of viable propagule acquisition. Pathogen distribution by adult shore flies was rapid over time at 2.21 cm² per hour per insect. The area over which the pathogen was distributed by adult shore flies increased with the increase in exposure time. The study showed that adult shore flies are efficient in the dispersal of the soilborne plant pathogen T. basicola.     

Quantification and correlation of vesicular-arbuscular mycorrhizal propagules with soil properties of some mollisols of northern India

The populations of vesicular-arbuscular mycorrhizae (VAM) propagules by the most probable number method in some mollisols and their correlations with some important soil properties were determined. On average, the six soils, Phoolbagh clay loam, Beni silty clay loam, Haldi loam, Nagla loam, Khamia sandy loam and Patherchatta sandy loam contained 4.9, 4.0, 7.9, 7.9, 3.3 and 13.0 propagules/g soil, respectively, i.e. none of the soils was found to be high in VAM. The size of the VAM population was compared to soil properties such as pH, organic carbon, sand content, available phosphorus and available potassium, cation-exchange capacity, silt and clay contents. A significant positive correlation (r=0.586) was only found with available soil phosphorus (P<0.05) and a significant negative correlation (r=-0.555) with soil clay content (P<0.05).Directorate research paper series No. 7862     

Fungi recovered from root-knot nematodes infecting vegetables under protected cultivation

A survey was conducted in root-knot nematode-infested plastic houses to determine the diversity and frequency of occurrence of fungi associated with the nematode. The relationships between percentage fungal parasitism and physicochemical properties of soil were also investigated. Fifty-nine plastic houses were sampled in southeastern Spain, 42 treated with nematicides and 17 left untreated. Eleven fungal genera and unidentified fungi were isolated from nematode eggs or juveniles. Fungal parasitism occurred more frequently in untreated (82.4%) than treated (50%) soils. The species richness in untreated soils ranged from 0 to 5, the Shannon–Wiener diversity index (a measurement of how many different fungi there are in site taking into account how evenly they are distributed among the site) from 0 to 2.01, and the evenness index from 0.46 to 0.99. In treated soils, species richness ranged from 0 to 4, the Shannon–Wiener diversity index from 0 to 1.61, and the evenness index from 0.81 to 1. Of the sites with nematophagous fungi, Arthrobotrys dactyloides (34%), Cylindrocarpon sp., Neosartoria hiratsukae (17%), and Fusarium solani (14%) were the fungi most frequently found. Physicochemical properties of soil were similar in nematicide treated and untreated soils. Percent fungal parasitism in untreated soils correlated positively with lime, silt and carbonate content of soil.     

Effects of soil pH and nitrogen fertility on the population dynamics of Thielaviopsis basicola

Black root rot of tobacco, caused by Thielaviopsis basicola, is generally severe at soil pH values >5.6 and suppressed under more acidic conditions (pH < 5.2). Soil acidifying fertilizers containing NH₄–N are generally recommended for burley tobacco production in North Carolina, but the effects of N form and application rate on development of black root rot and on the population dynamics of T. basicola have not been determined. Greenhouse and laboratory studies were conducted to evaluate the effects of N form (NH₄ ⁺ or NO₃ ^–) and rate on pathogen and disease parameters at several initial soil pH levels. A moderately-conducive field soil, initial pH 4.7, was adjusted to a pH of 5.5 or 6.5 by the addition of CaOH₂, then amended with the desired nitrogen form and rate. Pathogen populations were determined over time. In addition, spore production in extracts of roots from plants grown in the various nitrogen and pH treatments was determined. Finally, because tobacco responds to acidic soil conditions and exposure to NH₄–N by accumulating high concentrations of the polyamine putrescine, the toxicity of putrescine on vegetative growth and reproduction of T. basicola was investigated. Low soil pH and high levels of NH₄–N suppressed reproduction of T. basicola in soil and in root extract, while use of NO₃–N and depletion of NH₄–N resulted in rapid increases in populations of T. basicola. At 20 mM, putrescine inhibited hyphal growth by 60% and aleuriospore production by 98%. Fertilizers that reduced soil pH also reduced reproduction by T. basicola, and thus have potential for management of black root rot by suppressing populations of T. basicola over multiple years of crop production. The suppression of T. basicola and black root rot observed with NH₄–N amendments may partially be due to development of an inhibitory environment in the root and not solely to changes in rhizosphere pH.     

Evidence for ecological divergence across a mosaic of soil types in an Amazonian tropical tree: Protium subserratum (Burseraceae)

Soil heterogeneity is an important driver of divergent natural selection in plants. Neotropical forests have the highest tree diversity on earth, and frequently, soil specialist congeners are distributed parapatrically. While the role of edaphic heterogeneity in the origin and maintenance of tropical tree diversity is unknown, it has been posited that natural selection across the patchwork of soils in the Amazon rainforest is important in driving and maintaining tree diversity. We examined genetic and morphological differentiation among populations of the tropical tree Protium subserratum growing parapatrically on the mosaic of white‐sand, brown‐sand and clay soils found throughout western Amazonia. Nuclear microsatellites and leaf morphology were used to (i) quantify the extent of phenotypic and genetic divergence across habitat types, (ii) assess the importance of natural selection vs. drift in population divergence, (iii) determine the extent of hybridization and introgression across habitat types, (iv) estimate migration rates among populations. We found significant morphological variation correlated with soil type. Higher levels of genetic differentiation and lower migration rates were observed between adjacent populations found on different soil types than between geographically distant populations on the same soil type. P_ST‐F_ST comparisons indicate a role for natural selection in population divergence among soil types. A small number of hybrids were detected suggesting that gene flow among soil specialist populations may occur at low frequencies. Our results suggest that edaphic specialization has occurred multiple times in P. subserratum and that divergent natural selection across edaphic boundaries may be a general mechanism promoting and maintaining Amazonian tree diversity.     

Pathogenicity of four Phytophthora Species on Wild Cherry and Italian Alder Seedlings

Inoculation tests were carried out in the greenhouse on wild cherry (Prunus avium) and Italian alder (Alnus cordata) seedlings, to determine their susceptibility to certain Phytophthora species (P. citrophthora, P. alni, P. megasperma and P. cinnamomi) that are commonly present in the soil. Host susceptibility was evaluated in accordance with a disease index, with the lesion length after stem inoculation, and with a root system disease index. Wild cherry was found to be highly susceptible to P. citrophthora, and was also found to be susceptible to P. alni, although to a lesser extent. Italian alder was very susceptible to P. alni, but had only low susceptibility to P. citrophthora. The other Phytophthora species caused only modest symptoms. The danger to alder and wild cherry is all the greater because these trees not only share the same pathogens, but also commonly planted together in mixed stands. The results will now have to be confirmed by using a more natural inoculation method.     

Differences in Anatomical Structure and Lignin Content of Roots of pedunculate Oak and Wild Cherry-Tree Plantlets During Acclimation

The lignin contents and anatomical structure of roots of wild cherry (Prunus avium L.) and pedunculate oak (Quercus robur L.) plantlets were compared to explain differences in response during transfer from in vitro to ex vitro conditions. Lignification of cell walls increased significantly in both oak and cherry roots during the period of acclimation and finally lignin content of root tissues of in vitro propagated plantlets reached the levels not significantly different from seedlings grown in soil. Later on when secondary tissues appeared, lignified secondary xylem constituted most of the tissues of both species. The most conspicuous interspecific difference in root structure was the presence of phi-thickenings in cortical layers just outer to endodermis in cherry roots cultivated ex vitro. Formation of phi-thickenings was avoided in vitro and their presence thus seems to be under environmental control. Suberised well established exodermis was present in roots of oak but not detected in those of cherry. Very early development of exodermis in oak roots, preceding suberisation of endodermis, was recorded in vitro but not in well aerated soil. While multilayered and well-developed cork occurred in oak, only thin walled and less suberised secondary dermal tissues were found in cherry.     

The ability of Prunus avium× P. pseudocerasus `Colt' to form somatic embryos in vitro contrasts with the recalcitrance of P. avium

Somatic embryos were induced on roots excised from in vitro plants of Prunus avium× pseudocerasus `Colt'. On medium containing 6-benzylamino purine (BAP, 1.5 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D, 15 μM), a mean of 25 (s.e. ± 2.0) somatic embryos were produced on intact root systems and 15 (s.e. ± 1.7) on roots systems cut into 10 mm pieces. Most somatic embryos were formed directly on intact roots and indirectly (from callus) on sectioned roots. A mean of 2.5 (s.e. ± 0.25) secondary embryos per primary embryo were formed directly on primary embryos after they were transferred to medium containing BAP (1.5 μM), indole-3-butyric acid (10 μM) and 2,4-D (5 μM). After transfer to a medium containing BAP (2 μM) and gibberellic acid (GA<sub>3</sub>, 3 μM), shoots developed in 75% (s.e. ± 7.3) of the embryos. Somatic embryos were not induced on explants of in vitro roots or shoots of P. avium, and were induced infrequently on zygotic embryos, although a wide range of media were tested. Possible reasons for the contrasting embryogenic ability of `Colt' and P. avium are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

The influence of crop rotation and soil fumigation on a mycorrhizal fungal community associated with soybean

Population densities of mycorrhizal fungal propagules in a western Kentucky field highly productive for soybean were measured by bioassay throughout a soybean production season. The primary experimental variables were crop rotation (soybeans in 1985, then 2 years in corn, milo, fescue, or soybean, then soybean in 1988 on all plots when populations of propagules were determined) and soil fumigation with 67% methyl bromide/33% chloropicrin. Of the 20 species in three genera found, Glomus predominated both in terms of number of species and population densities. Most species of Glomus occurred at higher population densities in rotated plots than in continuous soybean plots. In continuous soybean plots, species of Gigaspora made up a much higher proportion of the mycorrhizal fungal community than in rotated crops. Species richness and diversity were lower, and dominance and equitability higher, in nonfumigated continuous soybean plots than in rotated plots early in the season, but the differences were not present at the end of the season. Soil fumigation killed most propagules in the upper 15 cm of soil, but after production of a crop of soybeans, populations of total propagules and most Glomus spp. recovered to prefumigation densities. However, Gigaspora margarita and Gigaspora gigantea did not recover similarly. Fumigation reduced species richness and diversity and increased dominance, but the effects were ameliorated by the end of the season. Colonization of roots was low during vegetative growth but increased rapidly after the onset of soybean reproduction. There was no evidence for mutualism during the early half of the season, perhaps due to high soil P and low dependency of soybean. Fumigation increased soybean yields. A stable mycorrhizal fungal community appeared to become established with continuous soybean production, and both crop rotation and soil fumigation disrupted the community.     

A cherry map from the inter-specific cross Prunus avium ‘Napoleon’ × P. nipponica based on microsatellite, gene-specific and isoenzyme markers

One hundred and sixty microsatellite (simple sequence repeat (SSR)) and six gene-specific markers revealing 174 loci were scored in 94 seedlings from the inter-specific cross of Prunus avium ‘Napoleon’ × Prunus nipponica accession F1292. The co-segregation data from these markers were used to construct a linkage map for cherry which spanned 680 cM over eight linkage groups with an average marker spacing of 3.9 cM per marker and just six gaps longer than 15 cM. Markers previously mapped in Prunus dulcis ‘Texas’ × Prunus persica ‘Earlygold’ allowed the cherry map to be anchored to the peach × almond map and showed the high level of synteny between the species. Eighty-four loci segregated in P. avium ‘Napoleon’ versus 159 in P. nipponica. The segregations of 32 isoenzyme loci in a subset of 47 seedlings from the progeny were scored, using polyacrylamide gel electrophoresis and/or isoelectric focusing separation followed by activity staining, and the co-segregation data were analysed along with those for 39 isoenzymes reported previously and for the 174 sequence-tagged site loci plus an additional two SSR loci. The second map incorporates 233 loci and spans 736 cM over eight linkage groups with an average marker spacing of 3.2 cM per marker and just two gaps greater than 15 cM. The microsatellite map will provide a useful tool for cherry breeding and marker-assisted selection and for synteny studies within Prunus; the gene-specific markers and isoenzymes will be useful for comparisons with maps of other rosaceous fruit crops. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.