Aptitude for mycorrhizal root colonization in Prunus rootstocks

Eighteen Prunus rootstock cultivars were inoculated with three arbuscular mycorrhizal fungi under greenhouse conditions in order to evaluate their affinity for mycorrhizal colonization. The rootstocks were peach–almond hybrids, peaches, plums and cherries of Spanish, French and Italian origin. Mycorrhizal colonization was low in plants inoculated with Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappe, and Glomus etunicatum Becker and Gerdemann. In contrast, Glomus intraradices Schenck and Smith, proved to be the most infective endophyte, achieving the highest mycorrhizal colonization rate in most of the rootstocks evaluated. Species of Prunus insititia L. were the only botanical group to show a consistently high affinity for mycorrhizal colonization with G. intraradices.     

Potential hazard characteristics of Tilia, Betula, and Acer trees removed in the Helsinki City Area during 2001–2003

In order to study decay, and to improve the management and protection of old urban trees, a total of 256 felled urban trees were examined during 2001–2003: 95 Tilia spp., 74 Betula spp., and 87 Acer spp. Most of the trees (73%) were located in the main parks and along the main streets in the downtown area of Helsinki City, Finland. The mean age of the trees was over 60 years, and the majority (64%) were old park trees. Poor condition and increasing risk of failure were the main reasons for felling in 82% of the cases. Thirty three percent of these trees were degenerated or dead, but the amenity value of 14% of the risk trees was still high. The latter were old, big trees which posed a potential hazard, but had a vital and balanced crown.Some characteristic profiles for potential failure were identified for each of the tree species studied: Ganoderma lipsiense in the butts and hollows in the stems of Tilia spp., weak fork formations together with Rigidoporus populinus on Acer spp., and degeneration together with decay in the stem on Betula spp.Decay fungi most commonly identified were R. populinus, G. lipsiense, Inonotus obliquus and Piptoporus betulinus. In addition, Kretzschmaria deusta was very common in three of the parks, and on every one of the tree species investigated.     

Selection of arbuscular mycorrhizal fungi for citrus growth promotion and Phytophthora suppression

In order to reduce unnecessary amount of P-fertilizer and severity of Phytophthora root rot in citrus orchards, the experiment was set up. Thirteen indigenous arbuscular mycorrhiza (AM) fungi species were isolated from rhizosphere soil of citrus orchards in Thailand and were then propagated into three host plants [sorghum (Sorghum bicolor), maize (Zea mays), and leek (Allium cepa)] by trap culture. We also tested whether indigenous AMF species (13 different species) could colonize into three cultivars of citrus scions and rootstocks (Shogun: Citrus reticulata Blanco cv. Shogun; Tangerine: C. reticulata; and C-35 citrange: Citrus sinensis × Poncirus trifoliata). With root colonization rates, the results indicated that Acaulospora tuberculata and Glomus etunicatum provided the best colonization in all citrus cultivars. We selected, therefore, those AMF species to verify their influences on citrus growth and Phytophthora root rot resistance. Three cultivars of citrus scions and rootstocks, Shogun, Tangerine and C-35 citrange, were inoculated with two effective indigenous AMF species, G. etunicatum or A. tuberculata in order to determine the influences on citrus growth. The plants were investigated to determine the mycorrhizal efficiency index (MEI), AM colonization, P content, and other parameters. Co-inoculation of AMF species (G. etunicatum or A. tuberculata) with Phytophthora nicotianae was also carried out in Shogun scion/C-35 citrange rootstocks. The results of citrus growth revealed that Shogun and Tangerine inoculated with G. etunicatum produced the highest MEI. Tangerine and C-35 citrange amended with fertilizers and G. etunicatum showed the highest P content in leaves. This indicated that G. etunicatum has an influence on citrus growth and P uptake, suggesting it to be the highly effective strain. Shogun scion/C-35 citrange rootstock combinations that were inoculated by both P. nicotianae and different AM fungi (G. etunicatum or A. tuberculata) showed root injury at low level of root rot symptom. However, the part of Shogun scions grafted on rootstocks showed severe symptom of shoot die back in treatment inoculated with P. nicotianae alone, while treatment inoculated with different AM species (G. etunicatum or A. tuberculata) and P. nicotianae rendered lower shoot die back symptoms than that of Phytophthora treatment. The low level of shoot die back symptom was shown at first, then healthy young shoot was restored. Our results indicated the facts that different host plants and different AMF species produced different outcomes of growth and pathogen resistance. The application of both AM isolates, therefore, has an enormous potential to be produced the inoculum for citrus orchards.     

Combining endophytic fungi and bacteria for the biocontrol of Radopholus similis (Cobb) Thorne and for effects on plant growth

Eight endophytic fungal and bacterial isolates with antagonistic activity against Radopholus similis were evaluated in vivo for their individual and combined effects on biocontrol of R. similis and on the growth of “Grand Naine” cultivar banana plantlets in the greenhouse. Penetration efficiency (PE) of R. similis was between 3 and 21% in 29 biological agents (BAs) treatments, less than the 29% of the nematode-alone control (p ≤ 0.0001); 24 of the BAs treatments did not differ from the PE of 5% for a nematicide control. Twenty nine BAs treatments exhibited antagonistic activity against nematodes which reduced final population levels between 18 and 93%, relative to those on nematode-alone control plants (p ≤ 0.0001), and 14 BAs treatments were statistically similar to the nematicide treatment (88% reduction). Twenty four BAs treatments had increments of plant root biomass ranging from 20 to 58%, greater than the control plants; 37% of the treatments with single and combined BAs inoculations had root length increments ranging from 29 to 54% compared with control and chemical treatment. The nematicide, Terbufos 10GR, did not affect plant growth.     

Effect of arbuscular mycorrhizal fungal inoculation on root system architecture of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings

Plant root system architecture is essential characteristics in relation to nutrient acquisition by root system from soil volume. Many environmental factors can affect the establishment of root system architecture, e.g. arbuscular mycorrhizal (AM) fungi. We inoculated the trifoliate orange (Poncirus trifoliata L. Raf.) seedlings with four AM fungal species in rhizoboxes, with non-inoculated seedlings as control. Using the WinRHIZO^® image analysis system, the root system architecture of seedlings was characterized. Results indicated that AM colonization did not affect the tap root length, the average root diameter, the basal root growth angle in spite that four AM fungal species exerted differential influence on the plant growth. Contrastingly, AM colonization significantly reduced the total root length, the root volume, the root surface area, but promoted the formation of lateral roots of high order. In addition, AM colonization induced more fine roots and less coarse roots. To our knowledge, it is the first report on the influence of AM fungi on the distribution of root diameter size classes. The mechanisms and implication of AM fungi on root system architecture is discussed.     

Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.)

Here we characterized eight novel polymorphic SSR markers, developed from an enriched genomic library of garlic (Allium sativum L.). These SSRs produced a total of 64 alleles across 90 garlic accessions, with an average of 8 alleles per locus. Values for observed (H_O) and expected (H_E) heterozygosity ranged from 0.16 to 0.77 (mean = 0.44) and from 0.22 to 0.86 (mean = 0.65), respectively. Six loci deviated significantly (P < 0.05) from Hardy–Weinberg equilibrium (HWE). The averages of gene diversity and PIC values were 0.65 and 0.62, respectively. The mean genetic similarity coefficient was 0.4380, indicating that among garlic accessions existed wide genetic variation. Based on 64 alleles obtained by 8 SSRs, a phenogram was constructed to understand the relationships among the 90 accessions. These newly developed SSRs should prove very useful tools for genotypes identification, assessment of genetic diversity and population structure in garlic.     

Role of bioinoculants in development of salt-resistant saplings of Morus alba (var. sujanpuri) in vivo

Pot experiments were conducted for the development of salt-resistant saplings of Morus alba (var. sujanpuri) involving bioinoculants, namely arbuscular mycorrhizal (AM) fungi, Azotobacter and indole butyric acid (IBA). The IBA and sodium chloride (NaCl) concentrations were optimized prior to the experiments. By using both low and high concentrations, 15 ppm IBA and 0.05% NaCl (w/v) were found to be optimum in acting synergistically with AM fungi and Azotobacter and also for increasing all the growth parameters and microbial count in the rhizosphere. For in vivo development of salt-resistant saplings, the optimal concentration of IBA, along with AM fungi and Azotobacter in different combinations, was applied. The saplings were irrigated regularly with 0.05% NaCl water. Although growth parameters such as AM infection percentage, AM spores per 100 g soil and Azotobacter cells/g soil were affected by NaCl watering, the inoculation of both bioinoculants significantly enhanced survival percentage of saplings from 25 to 50% under salt stress. Maximum survival (55%) of saplings was found with IBA (15 ppm)+AM fungi+Azotobacter.     

Effect of three Greek arbuscular mycorrhizal fungi in improving the growth,nutrient concentration,and production of essential oils of oregano and mint plants

Arbuscular mycorrhizal fungi have been widely used in agriculture to improve the cultivation of many crops. One of the aims of this study was the isolation and molecular identification of arbuscular mycorrhizal fungi isolated from mountain areas of Northern Greece (Ritini Pieria, Elatochori Pieria, Ambelakia Ossa). Only three isolates were obtained; two of Glomus etunicatum and one of G. lamellosum. The second objective of this study was to investigate the effect of these arbuscular mycorrhizal fungi on the concentration of macro- and micronutrients in tissues, the quantity and quality of essential oils and the growth of oregano and mint plants (two widely used aromatic plants in Greece). It was found that mycorrhizal oregano and mint plants had a higher content of essential oils and nutrient elements, and grew better than non-mycorrhizal plants. In addition, the composition of the essential oil in mycorrhizal plants differed from the oil of non-mycorrhizal plants. These results suggest that the use of mycorrhizal fungi may allow plant growth in low fertility soils, reduce fertilizer inputs and increase aromatic plant production of essential oils, They also indicate that it may be possible to use mycorrhizae to affect the quality of the essential oil produced.     

Selection of efficient arbuscular mycorrhizal fungi in the rhizosphere of cashew and their application in the cashew nursery

Mycorrhizal association in cashew (Anacardium occidentale L.) grown in five different plantation areas of South India was investigated. The soil samples were collected from all locations. Physico-chemical properties of rhizosphere soils, arbuscular mycorrhizal fungal spore count and root colonization were recorded. Species isolated were Acaulospora, Gigaspora, Glomus and Scutellospora. The mycorrhizal spore density was maximum at Thanjavur and minimum at Kudumiyamalai. Root colonization percentage oscillated between 20 and 30%. The species of Glomus, G. aggregatum Schenck and Smith, G. fasciculatum (Thaxter) Gerdemann and Trappe and G. mosseae (Nicol. and Gerd.) Gerdemann and Trappe were the most abundant in the majority of the experimental sites. Hence these three species were selected and used for artificial inoculation studies. Of the three mycorrhizal fungi G. fasciculatum significantly increased shoot length, internode number, internode length, number of leaves, stem diameter, root length and root number.     

Characterization of tetraploid plants regenerated via protoplast culture of Iris fulva and their crossability with Japanese irises

Characteristics such as flower form, size and color of outer and inner perianths, anthocyanins in outer perianths, size, color and fertility of pollen and self-fertility of diploid and tetraploid lines regenerated via protoplast culture of Iris fulva were examined and compared with those of the diploid wild line. Among these characteristics, flower form, inner and outer perianth sizes of the tetraploid lines were noticeable, because these lines had upward flower forms and bigger flowers than diploid lines. Furthermore, reciprocal crosses between diploid or tetraploid lines of I. fulva and I. ensata and those of I. fulva and I. laevigata were performed. Three seedlings were obtained from the cross of tetraploid I. fulva × diploid I. laevigata through embryo rescue. One of them was identified as the interspecific hybrid between tetraploid I. fulva and I. laevigata by flow cytometoric (FCM), cytological and molecular (RAPD) analyses. This is the first report on production of hybrids from these lines. I. fulva has unique brown flowers, and this trait could be very useful for flower color breeding of I. laevigata which lacks this color. Therefore, the hybrid of I. fulva (4×) × I. laevigata may be the best available gene source for brown color breeding of this species.     

Molecular identification and genetic analysis for 24 turf-type Cynodon cultivars by Sequence-Related Amplified Polymorphism markers

Bermudagrass (Cynodon ssp.) germplasm is genetically diverse and widely distributed in the world. The study was conducted to identify and assess the molecular variation and relationship among 24 cultivars developed in China, Australia and the USA. Sequence-Related Amplified Polymorphism (SRAP) was applied to cultivars identification in this study for the first time. Thirty of the 90 SRAP primer combinations generated a total of 274 clearly bands encompassing 249 (91%) polymorphic. Each bermudagrass cultivar has its unique binary code and can be distinguished from the others. Three distinct clusters were obtained by unweighted pair-group method with arithmetic averages based on the polymorphic markers. Coefficients of genetic distance among the genotypes ranged from 0.57 to 0.97. The results demonstrated that SRAP marker is a stable molecular marker technique for the identification of bermudagrass cultivars and their genetic relationships.     

Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits

Strawberry cultivation is not popular in Bangladesh due to the unpredictable climatic conditions and lack of proper cultivars. Using somaclonal variation, several new promising selections were generated and evaluated for their flowering and fruiting ability, adaptability and sustainability. To induce variation, plants were regenerated using various tissue culture techniques. Our results suggested that a high concentration of BAP in culture medium successfully resulted in the induction of somaclonal variation. Among the tissue culture techniques adopted in this study, meristem culture was most effective for induction of somaclonal variation. Twenty five putative somaclones with better horticultural features were subsequently selected and field evaluated for three clonal generations. Several of the selections reverted back to their original phenotype within 2–3 vegetative propagations. Three of the stable selections were distinct from each other in terms of fruit and other horticultural characters, and have potential for commercial cultivation in Bangladesh.     

Study of long-distance movement of Plum pox virus (Sharka) as an alternative resistance-evaluation method in Prunus

During the breeding programs for Plum pox virus (PPV, Sharka) resistance in Prunus, the evaluation of the new releases through symptoms observation on leaves has been contradictory and represents one of the main handicaps in these programs. In order to increase the accuracy of this traditional evaluation method, we here analyze an alternative method based on the study of the ability of a genotype to allow the long-distance movement of the virus through its vascular vessels. Two different plant models have been assayed: (a) in Model I, the inoculation was performed in the ‘GF305’ rootstock with a later grafting of the genotype under evaluation and a scion of healthy control ‘GF305’, to evaluate the long-distance movement through the studied genotype from the rootstock to the scion (xylem transport), and (b) in Model II, the inoculation with ‘GF305’ diseased scions was performed by grafting these diseased scions onto the studied genotypes, which were grafted onto healthy ‘GF305’ peach seedlings, to evaluate the long-distance movement through the studied genotype from the scion to the rootstock (phloem transport). The results show that, regardless of the presence of symptoms, susceptible genotypes allowed the movement of the virus through their vascular vessels in both directions studied. However, the resistant apricot ‘Stark Early Orange’ did not allow this movement. We propose the study of the ability of a genotype to allow the long-distance movement of the virus as an alternative and more accurate method for the evaluation of PPV resistance. However, this protocol is much more tedious than the traditional one and could be used mainly in the evaluation of a reduced number of more interesting genotypes.     

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species.     

Effects of an in vitro maturation treatment on plant recovery from avocado zygotic embryos

An efficient protocol for in vitro maturation of very immature, <10 mm, avocado embryos has been developed. The efficiency of plant recovery as well as the quality of the resulting plants was greatly improved by including a maturation phase prior to induction of germination. The influence of different factors, such as the gelling agent, organic supplements or abscisic acid, on embryo maturation and subsequent germination was tested. Optimum conditions were met when maturation was carried out in B5m medium supplemented with the Jensen's amino acids, an extra 88 mM sucrose and 6 g l⁻¹ agar as gelling agent. At these conditions, embryos which had been collected 68 days after pollination germinated at a 65% rate in solid medium, giving rise to healthy and vigorous plantlets. Anatomical differentiation and storage product accumulation occurring during the in vitro maturation phase were studied by means of histological techniques. Results obtained revealed that, at the end of the in vitro maturation period, embryos resembled the pattern previously established for avocado embryos matured under in vivo conditions: histodifferentiation had been accomplished and starch granules and protein bodies were abundant.     

In vitro micropropagation of the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck and effects of sprayed GA3 after transplantation to ex vitro conditions

We established the conditions to micropropagate the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck through axillary shoot development from isolated areoles. For the shoot proliferation stage different explant orientation (vertical and horizontal), type of cytokinin (BA, DAP and K), and concentrations (0, 1.25, 2,5, 5.0 and 7.5 mg/L) were evaluated. Media [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys. Plant. 15, 473–497: 50 and 100%], and carbohydrate concentration (0.25, 0.5, 0.75 and 1.0%) were studied to optimize individual shoot growth and elongation. Following micropropagation and plantlet acclimatization, the effects of GA₃ on plant growth were determined by spraying a series of increasing concentrations (0, 150, 300 and 450 ppm). A reliable and efficient protocol of micropropagation was established for this particular plant species. The greatest propagation ratio (shoot proliferation) was obtained when explants were cultured in vertical orientation (4.975 shoots per explant) as compared to horizontal position (3.692 shoots per explant). The addition of BA to the media resulted in increased shoot number per explant (8) in comparison to K and DA, which produced only 2 shoots in average. However, after 42 days of culture, significantly higher shoot length was obtained with DAP (14 mm) compared to K and BA (4 mm). After the shoot proliferation stage, an elongation subculture was performed prior rooting in which shoot growth was enhanced when crowns of shoots were cultured in 50% of basal salt formulation of Murashige and Skoog (1962) and low sucrose concentration (2.5 and 5%). Exogenous application of GA₃ after plantlet acclimatization on glasshouse conditions increased spine-hair (developed from areoles in young plants) length as part of short-term effects. However, significantly higher values were obtained in plantlets treated with 300 ppm of GA₃ when compared with the rest of the treatments. At the end of the study, the most important long-term effect produced by GA₃ was the suppression of total shoot growth. The micropropagation protocol described here and the conditions to grow the plants through fertigation plus the application of GA₃ that induced changes in the phenotype may be used in commercial exploitations to regenerate 12,500 plantelts in average after 12 months of culture and produce healthy plants with better ornamental characteristics and higher commercial value.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

Effect of arbuscular mycorrhizal fungi on growth,mineral nutrition,antioxidant enzymes activity and fruit yield of tomato grown under salinity stress

The purpose of this study was to investigate the mechanisms underlying alleviation of salt stress by arbuscular mycorrhizal fungi Glomus mosseae. Tomato (Lycopersicon esculentum L. cv. Zhongzha105) plants were cultivated in soil with 0, 50 and 100 mM NaCl. Mycorrhization alleviated salt induced reduction of root colonization, growth, leaf area, chlorophyll content, fruit fresh weight and fruit yield. The concentrations of P and K were higher in AM compared with nonAM plants grown under nonsaline and saline conditions. Na concentration was lower in AM than nonAM plants grown under nonsaline and saline conditions. AMF colonization was accompanied by an enhancement of activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) in leaves of both salt-affected and control plants. In addition, inoculation with AMF caused reduction in MDA content in comparison to salinized plants, indicating lower oxidative damage in the colonized plants.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.