Growth promotion and inhibition by antibiotic-producing fluorescent pseudomonads on citrus roots

Summary Forty-three strains of feeder root colonizing fluorescent pseudomonads from rough lemon (Citrus jambhiri Lush.) roots were examined for effects on rough lemon and sweet orange (Citrus sinensis Osbeck) seedlings. Plants inoculated with a single bacterial soil-drench had, after 10 months, a range of stimulatory (to 116%) and inhibitory effects (to 52%). Stimulatory bacteria particularly increased growth of root systems. Cultivar-specific inhibition and stimulation was evident in inoculations of rough lemon and sweet orange seedlings. Populations of fluorescent rhizobacteria on inoculated and noninoculated, as well as on stimulated and nonstimulated seedlings, did not differ significantly (10.8×10⁶ to 30.3×10⁶ CFU/g root). Population of fluorescent rhizobacteria on seedlings were higher than populations on feeder roots from grove trees (2.8 to 5.7×10⁶ CFU/g). Ninety-four and 81% of 251 fluorescent strains produced antibiotics against the fungusGeotrichum candidum and the bacteriumErwinia stewartii, respectively. Antibiotic activities of 90% of the antibiotic producing strains were repressed by Fe³⁺, indicating siderophore production. In comparison, only 9.6 and 15% of 94 randomly selected nonfluorescentPseudomonas strains were antibiotic producers. Differences between stimulatory and inhibitory or neutral bacteria were not apparent from antibiosis tests. On the basis of physiological tests,Pseudomonas putida was the most abundant (>62%) pseudomonad species on rough lemon roots. Growth stimulating strains appeared to be in bothP. putida andP. fluorescens groups. FewP. aeruginosa strains were identified on citrus roots.Florida Agricultural Experiment Stations Journal Series No.     

Effects of extracts of Alnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions

The amount of root mortality caused by root pathogens such as Phytophthora nicotianae (syn. Phytophthora parasitica) has typically been inferred from the net change in root length density in sequential soil cores. Because such measurements give information only on net changes in root populations, the actual rate of root turnover is often underestimated. We used minirhizotrons to track the fate of a large number of individual fine roots of mature field-grown citrus trees over a 6-month period. This method enabled us to examine the effect of P. nicotianae population levels on fine-root mortality. Seasonal and genotypic variation in patterns of citrus fine root mortality were associated with variation in population levels of P. nicotianae. Fine root lifespans were shorter when populations of P. nicotianae were high. Fine roots of the Phytophthora-susceptible rootstock, rough lemon (Citrus jamibhiri), had shorter median lifespans and supported larger populations of P. nicotianae than the fine roots of the more tolerant rootstock, Volkamer lemon (Citrus volkameriana). Rates of root mortality were either relatively constant for roots of all ages, or increased with age; the latter pattern was most pronounced for Volkamer lemon roots. Differences in the age-dependence of root mortality may, therefore, play a role in genotypic differences in tolerance of Phytophthora root rot by these two rootstocks. H Lambers Section editor     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Soil temperature and flooding effects on two species of citrus

Summary Rough lemon (Citrus jambhiri Lush.) and sour orange (C. aurantium L.) seedlings were grown at constant soil temperatures of 16, 24, and 33 C for 3 months. Shoot and root growth of rough lemon was greatest at 33 C while growth of sour orange was greatest at 24 C. There were no significant effects of soil temperature on shoot: root ratio, leaf water potential or stomatal conductance. The hydraulic conductivity of intact root systems of both species was highest when seedlings were grown at 16 C. Thus, acclimation through greater root conductivity at low soil temperature may have compensated for decreased root growth at 16 C and negated effects of soil temperature on plant water relations. Half the plants growing at each soil temperature were subsequently flooded. Within 1 week, the soil redox potential (Eh) dropped below zero mV, reaching a minimum Eh of –250mV after 3 weeks of flooded conditions. Flooded plants exhibited lower root conductivity, a cessation of shoot growth, lower leaf water potentials, lower stomatal conductances, and visual sloughing of fibrous roots. Decreases in root conductivity in response to flooding were large enough to account for the observed decreases in stomatal conductance.Florida Agricultural Experiment Stations Journal Series No. 4080.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Chemical composition and antimicrobial activity of Citrus essences on honeybee bacterial pathogen <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causal agent of American foulbrood

Antimicrobial properties and chemical composition of four citrus fruit essential oils to control Paenibacillus larvae, the causal agent of American foulbrood disease (AFB) were determined. This honeybee larvae disease occurs throughout the world and is found in many beekeeping areas of Argentina. Citrus fruit essential oils tested were those from grapefruit (Citrus paradisi), sweet orange (Citrus sinensis), mandarin (Citrus nobilis) and lemon (Citrus limon). The components of the essential oils were identified by SPME-GC/MS analysis. The antimicrobial activity of the oils against P. larvae were determined by the broth microdilution method. Two way ANOVA tests for minimum inhibitory concentrations (MICs) data and minimal bactericide concentrations (MBCs) data, indicated significant differences between the strains and the oils tested. The antimicrobial assays showed that the oil of C. paradisi inhibited the bacterial strains at the lowest concentrations tested, MICs and MBCs averages of 385.0 mg/l and 770.0 mg/l, respectively. This property could be attributed to the kind and percentage of the volatile components of the oil, like limonene (69.9%) and myrcene (9.6%). The use of essential oils or their specific volatile components individually against pests related to food provision may represent an alternative scope for the control of this serious disease because it does not leave toxic chemical residues in honey nor in its by products.     

Influence of Metalaxyl on Three Nematodes of Citrus

Metalaxyl significantly reduced population of Pratylenchus coffeae, Radopholus similis, and Tylenchulus semipenetrans in roots of Citrus limon (rough lemon) under greenhouse conditions. Postinoculation treatment of rough lemon seedlings was not as effective i n reducing nematode populations as was treatment before inoculation. Fewer nematodes infected metalaxyl-treated roots than nontreated roots. However, incubation of nematodes in metalaxyl did not inhibit nematode motility or their ability to locate and infect roots. Cellular responses to nematode injection differed between treated and nontreated tissues. Metalaxyl appeared to confer nematode contraol by modifying citrus roots such that a normally susceptible rootstock became tolerant.     

The use of the PMI/mannose selection system to recover transgenic sweet orange plants (<Emphasis Type="Italic">Citrus sinensis</Emphasis> L. Osbeck)

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.Abbreviations BAP Benzylaminopurine - CPR Chlorophenol red - EGTA Ethylene glycol-0-0- bis (2, aminoethyl) N, N, N, N tetraacetic acid - MTT [3-(4,5-Dimethyl thiazol-2-YL)-2,5-diphenyl] tetrazolium bromide - PMI Phosphomannose isomerase (EC 5.3.1.8) - PMS Phenazine methosulphate Communicated by L. Peña     

Antimicrobial proteins from cowpea root exudates: inhibitory activity against Fusarium oxysporum and purification of a chitinase-like protein

Plants exude a variety of substances through their external surfaces and from germinating seeds, some of which have an inhibitory action against plant pathogens. The aim of this study was the investigation and characterization of defense proteins present in exudates from roots of cowpea seedlings (Vigna unguiculata (L.) Walp.). Root exudates were collected from seedlings that were grown hydroponically in three different media, including, 100 mM sodium acetate buffer pH 4.5, water pH 6.0 and 100 mM sodium phosphate buffer pH 7.5. The proteins from these exudates were analyzed by SDS–PAGE and SDS–Tricine–PAGE and the presence of antimicrobial proteins in the exudates was investigated by immunological and enzymatic assays. Results showed that roots from cowpea seedlings contained -1,3-Glucanases, chitinases and lipid transfer proteins (LTPs), all of which may potentially function as plant defense proteins. Immunolocalization of one of these proteins, chitinase, revealed its presence in the xylem cell wall vessel elements. These exudates also demonstrated an inhibitory effect on the growth of the fungus, Fusarium oxysporum, in vitro. The results suggest that plant roots may exude a variety of proteins that may function to repress the growth of root pathogenic fungi.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees

Summary Iodoacetate-treated Citrus protoplasts from embryogenic nucellar calli of Sour orange (C. aurantium) or from Rough lemon (C. jambhiri) were fused with -irradiated protoplasts from a related genus, Microcitrus. The fused protoplasts were cultured to obtain colonies and micro-calli. Micro-calli derived from these two fusion combinations were isolated, propagated and differentiated into embryos, which subsequently regenerated trees having the morphology of Sour orange or Rough lemon. These intergeneric fusions resulted in mitochondria with novel DNA, indicating recombination between the chondriomes of Citrus and Microcitrus. Chloroplast DNA analyses of fusion-derived embryos indicated that they contained the chloroplasts of either fusion-partner or a mix of these chloroplasts. Later plastome analyses of leaves from fully differentiated plants showed that cybrids having Rough lemon morphology had either Rough lemon or Microcitrus chloroplast DNA, indicating complete sorting out of chloroplasts. Likewise, sorting out of Microcitrus chloroplasts was detected in a cybrid plant having Sour orange morphology.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel. No. 2663-E, 1989 series     

Water relations of rough lemon (Citrus jambhiri Lush.) citrus seedlings infected withFusarium solani

Summary Rough lemon citrus seedlings were inoculated withFusarium solani and evaluated for changes in water relations of leaves, stems, and roots. Inoculated seedlings had decreased leaf stomatal conductance, lower leaf water potential, lower water content, and higher leaf osmotic values compared to healthy plants. Visible wilt symptoms occurred as early as 24 h after inoculation. Transpiration and root conductivity were lower in diseased plants but stem conductivity in diseased plants did not differ from the control. Thus, wilting appears to be due to the inability of roots to supply water to the leaves.     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.     

Formation of Adventitious Buds in Decapitated Citrus Seedlings and the Effect of Some Growth Regulators

Young seedlings of various citrus species give rise, after decapitation,to adventitious buds at the cut end of the epicotyl. With suchtreatment, more buds formed on seedlings of sweet orange, sourorange, and grapefruit than of lemon, mandarin, and calamondin.Anatomical observations at the cut stem end show formation ofbuds occurring from tissues close to the cambium. Growth regulatorsapplied at the cut epicotyl end modified the patterns of budformation. 6-Benzylaminopurine (BAP) promoted bud formation;indole-3-butyric acid (IBA), abscisic acid (ABA), and gibberellicacid (GA₃) inhibited it. BAP reversed the inhibitory effectof GA₃. Gibberellic acid caused a typical elongation of thesprout axis. The use of decapitated seedlings as a tool forstudying stem differentiation in citrus is suggested.     

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa

A xylem-limited bacterium resemblingXylella fastidiosa has been shown previously by electron mmcroscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet organe tress in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth ofXylella fastidiosa. Bacterial colonies typical ofX. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7–10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4–3 m in length, and 0.2–0.4 m in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains ofX. fastidiosa. This result indicates that the CVC bacterium is a strain ofX. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless tress reacted negatively. Sweet organe seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic with 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.     

Rearing of Prays citri on callus derived from lemon stigma and style culture

A new method for rearing the citrus flower moth (Prays citri Mill.) (Lepidoptera, Yponomeutidae) on lemon [Citrus limon (L.) Burm.] callus is reported. In the present research callus (an undifferentiated mass of plant cells that can be grown under sterile conditions on an artificial medium in vitro) was induced from lemon stigma and style explants cultured on Murashige and Skoog (MS) medium supplemented with 500 mg l^–1 malt extract, 13.3 M 6-benzylaminopurine, and 146 mM sucrose. Also somatic embryogenesis and plant regeneration were obtained from the cultures of styles and stigmas of lemon. Adults were obtained from larvae on infested flowers collected in the field. Different oviposition substrates were used: white oval pearls (WOP), black oval pearls (BOP), rooted shoots (RS) of lemon obtained in vitro, and artificial flowers containing lemon callus (AF). Larvae were reared on lemon callus. Adults oviposited on RS, on WOP, and on AF. BOP were rejected as oviposition substrates. The flower moth reared on callus oviposited fertile eggs. In our tests P. citri completed three generations on callus or on callus plus shoots. In the latter case the larvae preferred callus and fed on shoots only after callus was completely eaten. The life cycle on callus at 23 ± 1 °C lasted about 21 days. There were significant differences between oviposition substrates for what concerns the number of eggs laid. It was observed that females generally preferred WOP (about 25 eggs/female) to AF (about 20 eggs/female) or RS (about 12 eggs/female) as oviposition substrate. Nevertheless the percentage of eggs that developed into adults was higher when AF sealed with stretched Parafilm were used (about 70% of eggs developed into adults). The method of rearing P. citri with AF was labour-saving and the feeding substrate (callus) had less tendency to become mouldy or decompose than when WOP and RS were used. Since such a diet is available for the insect all year round and callus can be produced in unlimited quantity, it could be possible to obtain a mass production of this moth.     

Genetic transformation of sweet orange with the coat protein gene of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> and evaluation of resistance against the virus

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.