Response of mycorrhizal grapevine to Armillaria mellea inoculation: disease development and polyamines

A study was conducted with the vine rootstock Richter 110 (Vitis berlandieri Planch. x Vitis rupestris L.) in order to assess whether the colonisation by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices (BEG 72) can delay the disease development in plants inoculated with the root-rot fungus Armillaria mellea (Vahl:Fr) Kummer, and to elucidate if the levels of polyamines (PAs) are modified in response to G. intraradices, A. mellea or by the dual infection. Four treatments were considered: control and G. intraradices-inoculated plants infected or not with A. mellea. Plant growth, mycorrhizal colonisation and disease development were monitored throughout the experiment. High performance liquid chromatography (HPLC) in combination with fluorescence spectrophotometry was used to separate and quantify free root and leaf polyamines. The slower development of pathogenic symptoms and the higher plant biomass of mycorrhizal plants inoculated with A. mellea indicate an increase of tolerance due to the AMF inoculation. The variations in free PA levels detected at the beginning of the pathogenic infection suggest that PAs may have a potential role in the signalling mechanisms of the tolerance of mycorrhizal plants against A. mellea.     

The effect of mycorrhizal infection on survival and growth renewal of micropropagated fruit rootstocks

Apple, peach and plum rootstocks were inoculated with the arbuscular mycorrhizal fungus Glomus sp. strain A6 on transplanting from in vitro to in vivo culture. The optimal root length for effective infection, assessed in apple rootstock M 25, was 0.1–1.5 cm, corresponding to the beginning of root elongation. When inoculated at this stage, plants showed maximal growth increase and survival. Mycorrhizal infection of the Mr. S. 2/5 rootstock induced earlier growth renewal after transplanting than in the controls. These results confirm previous reports that mycorrhizal inoculation, performed during transplantation from in vitro to in vivo culture, can enhance both the growth and the survival of plants.     

Evaluation of the biocidal effects of Brassica seed meal on Armillaria mellea

The antifungal properties of isothiocyanates released from the hydrolysis of the glucosinolate sinigrin were assessed on an Armillaria mellea strain in Petri dishes. The fumigation with sinigrin at 100 µM showed a fungicidal effect, while lower rates determined a temporary fungistatic effect. The application of increasing rates of solid and liquid biofumigant formulations on A. mellea inoculated potted peach plants showed an increase of soil nitrate concentration, basal respiration, microbial biomass, leaf nitrogen and chlorophyll concentration. These effects indicated a stimulating effect of Brassicaceae derivatives on soil biological activity and plant growth. The application of a rate of meal higher than 6.42 g kg^?1 soil caused a stunted growth and the death of some plants showing a clear phytotoxic effect of the treatments. The inhibition of A. mellea growth observed in in vitro trials was not evidenced in in vivo experiments due to the lack of infection symptoms in experimentally inoculated potted trees.     

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.     

Evaluating resistance of some rootstocks to grape phylloxera with in vitro and excised root testing systems

Summary The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Rul40, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Rul 40 rootstock was more susceptible than R99 and 3309C rootstocks.     

Studies on the mechanisms of barley seed deterioration

The type of chemical treatment used to control regrowth from hardwood stumps affects their colonization by fungi. Since such stumps often act as sources of infection for Armillaria mellea this may have important consequences. Ammonium sulphamate generally favours growth of species causing rapid decay, some of which compete well with A. mellea, whereas 2,4,5-trichlorophenoxyacetic acid tends to promote growth of Chondro-stereum purpureum which is replaced by A. mellea. The former treatment is recommended for situations where serious damage from A. mellea seems likely to occur, because the best hope of control is to ensure rapid killing of stump roots. Prompt colonization of stumps was obtained by inoculating the cut surface with spores of a wood-rotting fungus and then treating it with 40% ammonium sulphamate. In birch stumps jointly inoculated with Coriolus versicolor and A. mellea the amount of wood occupied by the latter after 4 yr was greatly reduced by comparison with controls inoculated with A. mellea alone. Stump inoculation might be worth developing for use in valuable plantations and other intensively managed areas such as parks and gardens, especially where measures such as stump excavation are impracticable.     

Response of Peach Seedlings to Infection by the Root Lesion Nematode Pratylenchus penetrans under Controlled Conditions

Twenty-one open pollinated populations of peach rootstock seedlings were evaluated for their response to infection by the root lesion nematode, Pratylenchus penetrans, over a period of 98 days. Nematode-infected peach seedling populations were shorter in plant height and had less shoot weight but more dry root weight than nematode-free controls. Rootstock differences were demonstrated for nematode increase over the 98-day period, and average total numbers of nematodes in soil and roots. Rootstocks were classified into three groups differing in total nematode population levels, ratio of nematode increase, and the number of nematodes per root. The heritable nature of rootstock response to nematodes was evident. Rootstocks showing the lowest response to nematode infection included Tzim Pee Tao, Rutgers Red Leaf, and two progenies of a cross of these two rootstocks.     

Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae

Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P ≤ 0.05) in plants infected by both parasites, regardless of inoculum density. Fibrous and tap root weights were 5% to 23% and 19% to 34% greater (P ≤ 0.05), respectively, in nematode-fungus combination treatments compared to the fungus alone. A second experiment was conducted, where plants were infected by the fungus, the nematode, both organisms, or neither organism. The soil mixture pH for 50% of the plants was adjusted from 4.5 to 7.0 to favor nematode infection. A higher rate of nematode infection of plants growing at pH 7.0 compared to pH 4.5 resulted in greater suppression of fungal development and greater inhibition of fungal damage to the plant. Compared to plants infected only by P. nicotianae, shoot and root weights were 37% and 33% greater (P ≤ 0.05), respectively, in plants infected by both parasites. These experiments have revealed antagonism between T. semipenetrans and P. nicotianae in citrus.     

Stimulation of Armillaria mellea by phenolic fungicides

Phenolic fungicides, which were initially fungicidal to mycelium of Armillaria mellea on the surface of well‐colonised wood billets, eventually stimulated the growth of A. mellea. An extensive growth of rhizomorphs was produced from A. mellea inoculum, which had been exposed to phenolic chemicals for 3 months, compared to few or no rhizomorphs produced from inoculum exposed to water or a suspension of a non‐phenolic fungicide, fenpropidin. Inoculated privet plants grown either in pots or under field conditions were treated with a range of fungicides; fenpropidin, phenyl phenol, cresylic acid or water (control) was applied every 6 months over 21/2 yr. Fenpropidin caused a slightly (but significantly) lower incidence of infection than occurred in untreated plants, but the phenolic fungicides, cresylic acid and phenyl phenol, did not reduce the incidence of infection. The severity of infection (% root circumference colonised at 5 cm depth) was greater following cresylic acid treatments than the other fungicides or water‐treated controls. Use of phenolic fungicides such as cresylic acid for the control of A. mellea may therefore be counter‐productive.     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan® real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical–chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.     

Response of grape root borer (Lepidoptera: Sesiidae) neonates to root extracts from Vitaceae species and rootstocks

Observations at regular intervals of the location of newly hatched grape root borer, Vitacea polistiformis (Harris), larvae moving freely within circular petri dish bioassays were used to measure and compare their response to dry filter paper discs treated with ethanol- or hexane-based extracts of roots from known and potential Vitaceae hosts and a nonhost. Larvae responded most strongly to discs treated with ethanol extracts, suggesting the presence of behaviorally active, polar compounds associated with roots. In single extract bioassays comparing extract versus solvent treated discs, larvae responded positively to ethanol extracts from all Vitis species and rootstocks and Virginia creeper [Parthenocissus quinquefolia (L.) Planch.], but not to apple (Malus domestica Borkh). Paired extract bioassays, in which an extract from the commercially important 3309 rootstock was used as the standard and presented simultaneously with extracts from other root sources, revealed examples of equal, significantly weaker and significantly stronger responses to the 3309 extract. Extracts of the 420 A and V. riparia 'Gloire' rootstocks appeared to possess qualities that elicited a consistently greater response than to 3309 extract in these pair-wise comparisons. The active compounds were eluted in ethanol during a 30-min extraction; larvae responded equally to 30- and 60-min 3309 root extracts in paired extract bioassays. Larvae responded equally to extracts of 3309 roots from three spatially separate vineyards in northern Virginia. These results are discussed in relation to the subterranean, plant-insect interactions of grape root borer neonates with the numerous native and non-native Vitis species that may serve as hosts in the eastern United States.     

Why does the bioluminescent fungus <Emphasis Type="Italic">Armillaria mellea</Emphasis> have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin—the key component of fungal bioluminescent system—was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.     

Impact of Plant Nutrition on Pratylenchus penetrans Infection of Prunus avium Rootstocks

A hypothesis that cherry rootstocks grown under optimal nutrient conditions are affected less by Pratylenchus penetrans infection than those grown under deficient nutrient conditions was tested by growing four Prunus avium L. rootstocks (''Mazzard'', ''Mahaleb'', ''GI148-1'', and ''GI148-8'') at a soil pH of 7.0 over a period of 3 months under greenhouse conditions (25 ñ 2 °C). Pratylenchus penetrans was inoculated at 0 (control) or 1,500 nematodes per g fresh root weight for a total of 3,600, 4,200, 10,500, and 11,400 per plant on Mazzard, Mahaleb, GI148-1, and GI148-8, respectively, with nutrients (commercial fertilizer) applied once at planting (deficient) or twice weekly (optimal). The experiment was repeated once. The optimum nutrient regime resulted in greater soil nutrient levels and plant growth; higher leaf concentrations of N, P, K, and Mg; and fewer P. penetrans than under the deficient nutrient regime. The addition of fertilizer either may increase nematode mortality in the soil or improve rootstock resistance to nematode infection. Increases in Ca in leaves from the nutrient-deficient and nematode-infected treatments suggested the plants were physiologically stressed. The Pf/Pi ratios indicated that these rootstocks may have had resistance to P. penetrans; however, because of the dominant role of nutrition in the experimental design, the question of resistance could not be properly addressed.     

Genetics of resistance to Meloidogyne incognita in crosses of grape rootstocks

Progeny testing was used to investigate the value of selected grape varieties as parents in breeding nematode-resistant rootstocks. Six pistillate-flowered rootstocks (Ramsey, Dog Ridge, Harmony, Freedom, 1613C, and 161-49C) and four staminate-flowered rootstocks (Riparia Gloire, 3309C, 1616C, and St. George) were used. Each male was crossed to each female. Six weeks after inoculation with 1,500 second-stage juveniles of Meloidogyne incognita race 3, roots were stained in an aqueous solution of eosin-Y (0.25 gm/l for 1 h). Seedling resistance was measured by counting the number of stained nematode egg masses visible per root system. Nematode reproduction on each cross was calculated as the average number of egg masses on ten seedlings per replicate. The females Harmony and Freedom produced the greatest level of resistance in their seedlings across all male parents. Seedlings of Dog Ridge, Ramsey, and 1613C had intermediate levels of resistance, while seedlings of 161-49C were the least resistant. The male 1616C contributed the greatest resistance to its progeny, while seedlings from crosses with the males Riparia Gloire, 3309C, and St. George had lower levels of resistance. Segregation ratios of resistant and susceptible seedlings are consistent with a single dominant allele model for root-knot nematode resistance.     

Growth,root colonization and nutrient status of Helianthemum sessiliflorum Desf. inoculated with a desert truffle Terfezia boudieri Chatin

This study aims to investigate the effects of inoculation using Terfezia boudieri Chatin ascospores (ectomycorrhizal fungus) on growth, root colonization and nutrient status of Helianthemum sessiliflorum Desf. seedlings grown in pots on two-soil types (gypseous and sandy loam). Mycorrhizal seedlings had significantly increased their height and leaf number compared to non-mycorrhizal ones. Regardless of mycorrhizal inoculation treatments, the plants growing on gypseous soil showed higher growth as compared to sandy loam one. It appears that inoculation with T. boudieri changed root morphology, increasing branching of first-order lateral roots of H. sessiliflorum seedlings. The highest root mycorrhizal colonization was recorded in inoculated seedlings on sandy loam soil (89%) when compared to gypseous one (52%). N, P and K concentrations in mycorrhizal seedlings were significantly improved by fungal inoculation. It can be concluded that inoculation of H. sessiliflorum with T. boudieri increased growth attributes and improved plant nutritional status.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Alleviation of salt stress in citrus seedlings inoculated with arbuscular mycorrhizal fungi depends on the rootstock salt tolerance

Seedlings of Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and Alemow (Citrus macrophylla Wester) were inoculated with a mixture of AM fungi (Rhizophagus irregularis and Funneliformis mosseae) (+AM), or left non-inoculated (−AM). From forty-five days after fungal inoculation onwards, half of +AM or −AM plants were irrigated with nutrient solution containing 50 mM NaCl. Three months later, AM significantly increased plant growth in both Cleopatra mandarin and Alemow rootstocks. Plant growth was higher in salinized +AM plants than in non-salinized −AM plants, demonstrating that AM compensates the growth limitations imposed by salinity. Whereas AM-inoculated Cleopatra mandarin seedlings had a very good response under saline treatment, inoculation in Alemow did not alleviate the negative effect of salinity. The beneficial effect of mycorrhization is unrelated with protection against the uptake of Na or Cl and the effect of AM on these ions did not explain the different response of rootstocks. This response was related with the nutritional status since our findings confirm that AM fungi can alter host responses to salinity stress, improving more the P, K, Fe and Cu plant nutrition in Cleopatra mandarin than in Alemow plants. AM inoculation under saline treatments also increased root Mg concentration but it was higher in Cleopatra mandarin than in Alemow. This could explain why AM fungus did not completely recovered chlorophyll concentrations in Alemow and consequently it had lower photosynthesis rate than control plants. AM fungi play an essential role in citrus rootstock growth and biomass production although the intensity of this response depends on the rootstock salinity tolerance.     

Cotton shoot plays a major role in mediating senescence induced by potassium deficiency

The objective of this study was to determine the roles of shoot and root in the regulation of premature leaf senescence induced by potassium (K) deficiency in cotton (Gossypium hirsutum L.). Two contrasting cultivars (CCRI41, more sensitive to K deficiency; and SCRC22, a less sensitive cultivar) were selected for self- and reciprocal-grafting, using standard grafting (one scion/one rootstock), Y grafting (two scions/one rootstock) and inverted Y grafting (one scion/two rootstocks) at the seedling stage. Standard grafting was studied in the field in 2007 and 2008. There were no obvious differences in senescence between CCRI41 and SCRC22 scions while supplied with sufficient K. However, SCRC22 scions showed significantly greater K content, SPAD values (chlorophyll content), soluble protein content and net photosynthetic rates than CCRI41 scions while grown in K deficient solution or soil, regardless of rootstock cultivars, grafting types, growth stage and growth conditions. Also, SCRC22 scions had greater yield and less variation in boll weight either between upper- and lower sympodials, or between proximal and distal fruit positions from the main stem in the field under K deficiency, probably owing to reduced leaf senescence. Although the effect of rootstocks on leaf senescence under K deficiency was significant in some cases, the scion cultivars explained the highest percentage of variations within grafting treatments. The shoot-to-root feedback signal(s), rather than high shoot demand for K nutrition, was involved in the shoot regulation of premature senescence in cotton plants, achieved possibly by altering root K uptake.