Common mycorrhizal networks activate salicylic acid defense responses of trifoliate orange (Poncirus trifoliata).

Citrus canker, caused by Xanthomonas axonopodis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and further affect their disease resistance, possibly by modulating the activity of salicylic acid (SA), a key phytohormone in disease resistance. Common mycorrhizal networks (CMNs) can interconnect plants, but it is not yet clear whether CMNs promote resistance to citrus canker and, if so, whether SA signaling is involved in this process. To test this possibility, we used a two-chambered rootbox to establish CMNs between trifoliate orange (Poncirus trifoliata) seedlings in chambers inoculated (treated) or not (neighboring) with the AMF, Paraglomus occultum. A subset of the AMF-inoculated seedlings were also inoculated with Xac (+AMF+Xac). At 2 d post-inoculation (dpi), compared with the +AMF-Xac treatment, neighboring seedlings in +AMF+Xac treatment had lower expression levels of the SA biosynthetic genes, PtPAL, PtEPS1, and PtPBS3, but higher SA levels, which attributed to the upregulation of PtPAL and PtPBS3 in treated seedlings and the transfer of SA, via CMNs, to the neighboring seedlings. At 4 dpi, the pathogenesis-related (PR) protein genes, PtPR1, PtPR4, and PtPR5, and the transcriptional regulatory factor gene, PtNPR1, were activated in neighboring seedlings of +AMF+Xac treatment. At 9 dpi, root phenylalanine ammonia-lyase activity and total soluble phenol and lignin concentrations increased in neighboring seedlings of +AMF+Xac treatment, likely due to the linkage and signal transfer, via CMNs. These findings support the hypothesis that CMNs transfer the SA signal from infected to neighboring healthy seedlings, to activate defense responses and affording protection to neighboring plants against citrus canker infection.

Molecular Basis of Citrus sunki Susceptibility and Poncirus trifoliata Resistance Upon Phytophthora parasitica Attack.

Coevolution has shaped the molecular basis of an extensive number of defense mechanisms in plant-pathogen interactions. Phytophthora parasitica, a hemibiothrophic oomycete pathogen and the causal agent of citrus root rot and gummosis, interacts differently with Citrus sunki and Poncirus trifoliata, two commonly favored citrus rootstocks that are recognized as susceptible and resistant, respectively, to P. parasitica. The molecular core of these interactions remains elusive. Here, we provide evidence on the defense strategies employed by both susceptible and resistant citrus rootstocks, in parallel with P. parasitica deployment of effectors. Time course expression analysis (quantitative real-time polymerase chain reaction) of several defense-related genes were evaluated during i) plant disease development, ii) necrosis, and iii) pathogen effector gene expression. In C. sunki, P. parasitica deploys effectors, including elicitins, NPP1 (necrosis-inducing Phytophthora protein 1), CBEL (cellulose-binding elicitor and lectin activity), RxLR, and CRN (crinkler), and, consequently, this susceptible plant activates its main defense signaling pathways that result in the hypersensitive response and necrosis. Despite the strong plant-defense response, it fails to withstand P. parasitica invasion, confirming its hemibiothrophic lifestyle. In Poncirus trifoliata, the effectors were strongly expressed, nevertheless failing to induce any immunity manipulation and disease development, suggesting a nonhost resistance type, in which the plant relies on preformed biochemical and anatomical barriers.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

Mycorrhiza alters the profile of root hairs in trifoliate orange.

Root hairs and arbuscular mycorrhiza (AM) coexist in root systems for nutrient and water absorption, but the relation between AM and root hairs is poorly known. A pot study was performed to evaluate the effects of four different AM fungi (AMF), namely, Claroideoglomus etunicatum, Diversispora versiformis, Funneliformis mosseae, and Rhizophagus intraradices on root hair development in trifoliate orange (Poncirus trifoliata) seedlings grown in sand. Mycorrhizal seedlings showed significantly higher root hair density than non-mycorrhizal seedlings, irrespective of AMF species. AMF inoculation generally significantly decreased root hair length in the first- and second-order lateral roots but increased it in the third- and fourth-order lateral roots. AMF colonization induced diverse responses in root hair diameter of different order lateral roots. Considerably greater concentrations of phosphorus (P), nitric oxide (NO), glucose, sucrose, indole-3-acetic acid (IAA), and methyl jasmonate (MeJA) were found in roots of AM seedlings than in non-AM seedlings. Levels of P, NO, carbohydrates, IAA, and MeJA in roots were correlated with AM formation and root hair development. These results suggest that AMF could alter the profile of root hairs in trifoliate orange through modulation of physiological activities. F. mosseae, which had the greatest positive effects, could represent an efficient AM fungus for increasing fruit yields or decreasing fertilizer inputs in citrus production.

Community analysis of arbuscular mycorrhizal fungi in roots of Poncirus trifoliata and Citrus reticulata based on SSU rDNA.

Morphological observation of arbuscular mycorrhizal fungi (AMF) species in rhizospheric soil could not accurately reflect the actual AMF colonizing status in roots, while molecular identification of indigenous AMF colonizing citrus rootstocks at present was rare in China. In our study, community of AMF colonizing trifoliate orange (Poncirus trifoliata L. Raf.) and red tangerine (Citrus reticulata Blanco) were analyzed based on small subunit of ribosomal DNA genes. Morphological observation showed that arbuscular mycorrhizal (AM) colonization, spore density, and hyphal length did not differ significantly between two rootstocks. Phylogenetic analysis showed that 173 screened AMF sequences clustered in at least 10 discrete groups (GLO1~GLO10), all belonging to the genus of Glomus Sensu Lato. Among them, GLO1 clade (clustering with uncultured Glomus) accounting for 54.43% clones was the most common in trifoliate orange roots, while GLO6 clade (clustering with Glomus intraradices) accounting for 35.00% clones was the most common in red tangerine roots. Although, Shannon-Wiener indices exhibited no notable differences between both rootstocks, relative proportions of observed clades analysis revealed that composition of AMF communities colonizing two rootstocks varied severely. The results indicated that native AMF species in citrus rhizosphere had diverse colonization potential between two different rootstocks in the present orchards.

Wide-ranging transcriptomic analysis of Poncirus trifoliata, Citrus sunki, Citrus sinensis and contrasting hybrids reveals HLB tolerance mechanisms.

Huanglongbing (HLB), caused mainly by 'Candidatus Liberibacter asiaticus' (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

[Effects of arbuscular mycorrhizal fungi on different iron species in Poncirus trifoliata rhizospheric soil].

OBJECTIVE: We studied the effects of arbuscular mycorrhizal (AM) fungus (Glomus versiforme) on iron species at different trifoliate orange [Poncirus trifoliata (L.) Raf.] rhizospheric soil with pot culture. METHODS: The soil range in horizon was separated into 0 - 2 cm, 2 - 4 cm and 4 - 8 cm away from the citrus taproot by nylon bags with 32 microm sieve, whereby citrus roots could not across but AM hyphae could. Mycorrhizal colonization was confirmed by trypan blue staining method. Iron species contents were quantified by atomic spectrometry, and phosphorus contents was through phospho-vanado- molybdate colorimetry. RESULTS: Available iron contents in AM treatment were followed the order by 0- 2 cm > 2 -4 cm > 4 - 8 cm. AM fungus decreased the contents of Exch-Fe, OM-Fe, RES-Fe. The concentration of Exch-Fe, Carb-Fe and MnOX-Fe became zero one year later. AM colonization was significantly positive relation with RES-Fe (P < 0.01) CONCLUSION: Arbuscular mycorrhizal fungi could affect the activation on mineral elements, and improve the available iron contents through the changes on iron species in soil.

[Effects of arbuscular mycorrhizal fungi on growth and iron uptake of Poncirus trifoliata under different pH].

OBJECTIVE: We studied the effects of arbuscular mycorrhizal fungus ( Glomus versiforme) on growth and iron uptake of trifoliate orange [Poncirus trifoliata (L.) Raf.] at different pH levels of nutrient solution. METHODS: P. trifoliata seedlings were grown in substrates watered by nutrient solution with 50 microM Feethylenediaminetetraacetic acid (EDTA) at pH values of 5.0, 6.0 (as control), 7.0 and 8.0 with a sand culture. Mycorrhizal colonization was tested by trypan blue staining method. Chlorophyll concentratration and root Fe (III) chelate reductase activity were determined by spectrophotometer. Potassium and active iron contents were quantified by atomic spectrometry, and phosphorus contents was through phospho-vanado- molybdate colorimetry. RESULTS: The colonization of Glomus versiforme significantly increased the plant height, stem diameter, leaf numbers, and dry mass. Arbuscular mycorrhizal fungus significantly enhanced the accumulation of chlorophyll, active iron, total iron and root Fe(III) chelate reductase activity, and decreased P/Fe and 50 (10P + K)/Fe ratios. All biomass, iron contents and root Fe(III) chelate reductase activities of P. trifoliata seedlings at pH 6.0 level were the maximum both in inoculated and non-inoculated treatments. CONCLUSION: Arbuscular mycorrhizal fungi could remedy chlorosis caused by iron-deficiency in citrus, and 6.0 was the optimal pH value for the growth of P. trifoliata seedlings.

Mycorrhizal symbiosis down-regulates or does not change root aquaporin expression in trifoliate orange under drought stress.

Arbuscular mycorrhizas absorb water from soil to host plants, while the relationship between mycorrhizas and aquaporins (AQPs, membrane water channel proteins, which function in water transport) in mycorrhizal plants is unclear. In this study, Funneliformis mosseae-colonized trifoliate orange (Poncirus trifoliata) seedlings were grown in pots fitted with 37-µm nylon meshes at the bottom of each pot to allow mycorrhizal hyphae absorb water from an outer beaker. The expression of seven plasma membrane intrinsic proteins (PIPs) genes, six tonoplast intrinsic proteins (TIPs) genes, and four nodulin-26 like intrinsic proteins (NIPs) genes were analyzed in roots of both well-watered (WW) and drought stressed (DS) plants. The six-week DS plants dramatically increased hyphal water absorption rate by 1.4 times, as compared with WW plants. Mycorrhizal plants exhibited greater plant growth performance, leaf water status (water potential and relative water content), and gas exchange under both WW and DS conditions. Mycorrhizal inoculation induced diverse expression patterns in these AQPs under WW: up-regulation of PtNIP1;1, PtPIP2;1, and PtPIP2;5, down-regulation of PtNIP1;2, PtNIP6;1, PtPIP1;2, PtPIP1;5, PtPIP2;8, PtTIP1;1, PtTIP1;2, PtTIP1;3, and PtTIP5;1, and no changes in other AQPs. However, the expression of PtPIPs and PtNIPs was down-regulated by mycorrhizal inoculation under DS, and PtTIPs was not induced by mycorrhizal colonization under DS. The expression pattern of AQPs in response to mycorrhizas under DS is a way of mycorrhizal plants to minimize water loss.

Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress.

Root-hair growth and development regulated by soil microbes is associated with auxin. In this background, we hypothesized that mycorrhizal fungal inoculation induces greater root-hair growth through stimulated auxin synthesis and transport under water stress conditions. Trifoliate orange (Poncirus trifoliata) was inoculated with an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) under well-watered (WW) and drought stress (DS) for 9 weeks. Compared with non-AM seedlings, AM seedlings displayed significantly higher density, length, and diameter of root hairs and root indoleacetic acid (IAA) level, whereas lower total root IAA efflux, regardless of soil moisture status. Root PtYUC3 and PtYUC8 involved in IAA biosynthesis were up-regulated by mycorrhization under WW and DS, whereas AM-modulated expression in PtTAA1, PtTAR2, PtYUC4, and PtYUC6 depended on status of soil moisture. Mycorrhizal inoculation down-regulated the transcript level of root auxin efflux carriers like PtPIN1 and PtPIN3, whereas significantly up-regulated the expression of root auxin-species influx carriers like PtABCB19 and PtLAX2 under DS. These results indicated that AMF-stimulated greater root-hair growth of trifoliate orange under DS that is independent on AMF species is related with mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to enhance drought tolerance.

Alleviation of drought stress by mycorrhizas is related to increased root H2O2 efflux in trifoliate orange.

The Non-invasive Micro-test Technique (NMT) is used to measure dynamic changes of specific ions/molecules non-invasively, but information about hydrogen peroxide (H2O2) fluxes in different classes of roots by mycorrhiza is scarce in terms of NMT. Effects of Funneliformis mosseae on plant growth, H2O2, superoxide radical (O2·-), malondialdehyde (MDA) concentrations, and H2O2 fluxes in the taproot (TR) and lateral roots (LRs) of trifoliate orange seedlings under well-watered (WW) and drought stress (DS) conditions were studied. DS strongly inhibited mycorrhizal colonization in the TR and LRs, whereas mycorrhizal inoculation significantly promoted plant growth and biomass production. H2O2, O2·-, and MDA concentrations in leaves and roots were dramatically lower in mycorrhizal seedlings than in non-mycorrhizal seedlings under DS. Compared with non-mycorrhizal seedlings, mycorrhizal seedlings had relatively higher net root H2O2 effluxes in the TR and LRs especially under WW, as well as significantly higher total root H2O2 effluxes in the TR and LRs under WW and DS. Total root H2O2 effluxes were significantly positively correlated with root colonization but negatively with root H2O2 and MDA concentrations. It suggested that mycorrhizas induces more H2O2 effluxes of the TR and LRs, thus, alleviating oxidative damage of DS in the host plant.

Mycorrhizal trifoliate orange has greater root adaptation of morphology and phytohormones in response to drought stress.

Plant roots are the first parts of plants to face drought stress (DS), and thus root modification is important for plants to adapt to drought. We hypothesized that the roots of arbuscular mycorrhizal (AM) plants exhibit better adaptation in terms of morphology and phytohormones under DS. Trifoliate orange seedlings inoculated with Diversispora versiformis were subjected to well-watered (WW) and DS conditions for 6 weeks. AM seedlings exhibited better growth performance and significantly greater number of 1st, 2nd, and 3rd order lateral roots, root length, area, average diameter, volume, tips, forks, and crossings than non-AM seedlings under both WW and DS conditions. AM fungal inoculation considerably increased root hair density under both WW and DS and root hair length under DS, while dramatically decreased root hair length under WW but there was no change in root hair diameter. AM plants had greater concentrations of indole-3-acetic acid, methyl jasmonate, nitric oxide, and calmodulin in roots, which were significantly correlated with changes in root morphology. These results support the hypothesis that AM plants show superior adaptation in root morphology under DS that is potentially associated with indole-3-acetic acid, methyl jasmonate, nitric oxide, and calmodulin levels.

Mycorrhizas alter sucrose and proline metabolism in trifoliate orange exposed to drought stress.

Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in plants, whereas little is known regarding AMF contribution to sucrose and proline metabolisms under drought stress (DS). In this study, Funneliformis mosseae and Paraglomus occultum were inoculated into trifoliate orange (Poncirus trifoliata) under well watered and DS. Although the 71-days DS notably (P < 0.05) inhibited mycorrhizal colonization, AMF seedlings showed significantly (P < 0.05) higher plant growth performance and leaf relative water content, regardless of soil water status. AMF inoculation significantly (P < 0.05) increased leaf sucrose, glucose and fructose concentration under DS, accompanied with a significant increase of leaf sucrose phosphate synthase, neutral invertase, and net activity of sucrose-metabolized enzymes and a decrease in leaf acid invertase and sucrose synthase activity. AMF inoculation produced no change in leaf ornithine-δ-aminotransferase activity, but significantly (P < 0.05) increased leaf proline dehydrogenase activity and significantly (P < 0.05) decreased leaf both Δ1-pyrroline-5-carboxylate reductase and Δ1-pyrroline-5-carboxylate synthetase activity, resulting in lower proline accumulation in AMF plants under DS. Our results therefore suggest that AMF strongly altered leaf sucrose and proline metabolism through regulating sucrose- and proline-metabolized enzyme activities, which is important for osmotic adjustment of the host plant.

Effects of Common Mycorrhizal Network on Plant Carbohydrates and Soil Properties in Trifoliate Orange-White Clover Association.

Common mycorrhizal network (CMN) allows nutrients and signals to pass between two or more plants. In this study, trifoliate orange (Poncirus trifoliata) and white clover (Trifolium repens) were planted in a two-compartmented rootbox, separated by a 37-µm nylon mesh and then inoculated with an arbuscular mycorrhizal fungus (AMF), Diversispora spurca. Inoculation with D. spurca resulted in formation of a CMN between trifoliate orange and white clover, whilst the best AM colonization occurred in the donor trifoliate orange-receptor white clover association. In the trifoliate orange-white clover association, the mycorrhizal colonization of receptor plant by extraradical hyphae originated from the donor plant significantly increased shoot and root fresh weight and chlorophyll concentration of the receptor plant. Enzymatic activity of soil ß-glucoside hydrolase, protease, acid and neutral phosphatase, water-stable aggregate percentage at 2-4 and 0.5-1 mm size, and mean weight diameter in the rhizosphere of the receptor plant also increased. The hyphae of CMN released more easily-extractable glomalin-related soil protein and total glomalin-related soil protein into the receptor rhizosphere, which represented a significantly positive correlation with aggregate stability. AMF inoculation exhibited diverse changes in leaf and root sucrose concentration in the donor plant, and AM colonization by CMN conferred a significant increase of root glucose in the receptor plant. These results suggested that CMN formed in the trifoliate orange-white clover association, and root AM colonization by CMN promoted plant growth, root glucose accumulation, and rhizospheric soil properties in the receptor plant.

[Effects of arbuscular mycorrhizal fungi on root system morphology and sucrose and glucose contents of Poncirus trifoliata].

The effects of inoculation with Glomus mosseae, G. versiforme, and their mixture on plant growth, root system morphology, and sucrose and glucose contents of trifoliate orange (Poncirus trifoliata L.) were studied by pot culture. The results showed that all the inoculated treatments significantly increased the plant height, stem diameter, leaf number, and shoot and root biomass. In addition, the mycorrhizal treatments significantly increased the number of 1st, 2nd, and 3rd lateral roots. Inoculation with arbuscular mycorrhizal fungi significantly increased the root projected area, surface area, volume, and total root length (mainly 0-1 cm root length), but decreased the root average diameter. Meanwhile, G. versiforme showed the best effects. Mycorrhizal inoculation significantly increased the leaf sucrose and root glucose contents, but decreased the leaf glucose and root sucrose contents. Owing to the 'mycorrhizal carbon pool' in roots, inoculation with arbuscular mycorrhizal fungi resulted in high glucose content and low sucrose content of roots, which would facilitate the root growth and development, thereby the establishment of better root system morphology of host plants.

Direct and indirect effects of glomalin, mycorrhizal hyphae, and roots on aggregate stability in rhizosphere of trifoliate orange.

To test direct and indirect effects of glomalin, mycorrhizal hyphae, and roots on aggregate stability, perspex pots separated by 37-µm nylon mesh in the middle were used to form root-free hyphae and root/hyphae chambers, where trifoliate orange (Poncirus trifoliata) seedlings were colonized by Funneliformis mosseae or Paraglomus occultum in the root/hyphae chamber. Both fungal species induced significantly higher plant growth, root total length, easily-extractable glomalin-related soil protein (EE-GRSP) and total GRSP (T-GRSP), and mean weight diameter (an aggregate stability indicator). The Pearson correlation showed that root colonization or soil hyphal length significantly positively correlated with EE-GRSP, difficultly-extractable GRSP (DE-GRSP), T-GRSP, and water-stable aggregates in 2.00-4.00, 0.50-1.00, and 0.25-0.50 mm size fractions. The path analysis indicated that in the root/hyphae chamber, aggregate stability derived from a direct effect of root colonization, EE-GRSP or DE-GRSP. Meanwhile, the direct effect was stronger by EE-GRSP or DE-GRSP than by mycorrhizal colonization. In the root-free hyphae chamber, mycorrhizal-mediated aggregate stability was due to total effect but not direct effect of soil hyphal length, EE-GRSP and T-GRSP. Our results suggest that GRSP among these tested factors may be the primary contributor to aggregate stability in the citrus rhizosphere.

Mycorrhizal-mediated lower proline accumulation in Poncirus trifoliata under water deficit derives from the integration of inhibition of proline synthesis with increase of proline degradation.

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵(1)-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.

Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus.

Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been observed in the genus Poncirus and some of its hybrids. This study compares transcriptional changes in tolerant US-897 (Citrus reticulata Blanco×Poncirus trifoliata L. Raf.) and susceptible 'Cleopatra' mandarin (C. reticulata) seedlings in response to infection with Las using the Affymetrix GeneChip citrus array, with the main objective of identifying genes associated with tolerance to HLB. Microarray analysis identified 326 genes which were significantly upregulated by at least 4-fold in the susceptible genotype, compared with only 17 genes in US-897. Exclusively upregulated in US-897 was a gene for a 2-oxoglutarate (2OG) and Fe(II)-dependant oxygenase, an important enzyme involved in the biosynthesis of plant secondary metabolites. More than eight hundred genes were expressed at much higher levels in US-897 independent of infection with Las. Among these, genes for a constitutive disease resistance protein (CDR1) were notable. The possible involvement of these and other detected genes in tolerance to HLB and their possible use for biotechnology are discussed.

[Effects of inoculation with arbuscular mycorrhizal fungi on AIPO4 uptake by Poncirus trifoliata].

With sand culture in greenhouse, this paper studied the effects of inoculation with arbuscular mycorrhizal (AM) fungi Glomus mosseae and G. versiforme on the utilization of insoluble phosphate AlPO4 (Al-P) by trifoliate orange (Poncirus trifoliata) seedlings. The results indicated that inoculation with AM fungi notably increased the dry matter, P content, and P absorption rate of P. trifoliata. The contribution of mycorrhiza, P uptake by plant and AM fungi, and the contribution of AM fungi in P uptake by P. trifoliata increased greatly with increasing Al-P level in the medium. Inoculation with AM fungi greatly increased the excretion of phosphatase, especially acid- and neutral phosphatase, from plant root and hyphae, but the excretion amount decreased with increasing Al-P level. The contribution of the fungi in P uptake by P. trifoliata was significantly positively correlated with the amounts of acid-, neutral-, alkaline-, and total phosphatase.

[Effects of arbuscular mycorrhiza on drought tolerance of Poncirus trifoliata].

This paper evaluated the effects of arbuscular mycorrhizal colonization on the growth and drought tolerance of Poncirus trifoliata in a potted culture under natural water stress and resuming water supply. The results showed that the arbuscular mycorrhizal colonization by Glomus mosseae 93 could significantly increase the plant height, stem diameter, and fresh weight of myocrrhizal plant. By the end of the experiment, the survival percentage of transplanted mycorrhizal plant was 7.1% higher than that of nonmyocrrhizal plant. During natural water stress and resuming water supply, the soluble sugar and soluble protein contents and the SOD, POD and CAT activities of root or leaf were higher or significantly higher in mycorrhizal than in nonmycorrhizal plant, suggesting that arbuscular mycorrhizal colonization could improve the osmoregulatory response of myocrrhizal plant, enhance its protective capability, and depress its cell membrane oxidative damage to lipids. Consequently, the drought tolerance of inoculated Poncirus trifoliata was enhanced. It was considered that the functional mechanism of arbuscular mycorrhizal fungus in enhancing the drought tolerance of host plant was closely related to the protective systems such as SOD, POD, CAT, and soluble protein, etc.