Design, Development, and Use of Molecular Primers and Probes for the Detection of Gluconacetobacter Species in the Pink Sugarcane Mealybug

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of “squashed” whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.     

Acetic Acid Bacterial Biota of the Pink Sugar Cane Mealybug, Saccharococcus sacchari, and Its Environs

Saccharococcus sacchari is the primary colonizer of the developing “sterile” tissue between the leaf sheath and stem of sugar cane. The honeydew secreted by the mealybugs is acidic (about pH 3) and supports an atypical epiphytic microbiota dominated by acetobacter-like bacteria and acidophilic yeast species. However, Erwinia and Leuconostoc species predominate within the leaf sheath pocket region when the mealybugs die out. The unidentified acetobacters were readily isolated from S. sacchari throughout its life cycle and from other genera of mealybugs on sugar cane and various other plants, both above and below ground. No other insect present on sugar cane was a significant vector of acetic acid bacteria. The major factors restricting microbial diversity within the environs of mealybugs were considered to be yeast activity along with bacterial production of acetic acid, ketogluconic acids, and gamma-pyrones, in association with their lowering of pH. The microbial products may aid in suppressing the attack by the parasitic mold Aspergillus parasiticus on mealybugs but could act as attractants for the predatory fruit fly Cacoxenus perspicax.     

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchariCockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarum L.). In this study, bothGlac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated withGlac. sacchari.Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.     

Low recovery frequency of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> from plants and associated mealybugs in Cuban sugarcane fields

This study was aimed to isolate and identify the N₂-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N₂-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.     

一株高效甘蔗内生固氮菌NN08200的鉴定及其对甘蔗的促生长作用

【背景】生产上过高的氮肥投入是我国农业可持续发展的重要限制因子之一。利用生物固氮是减少氮肥施用量最为有效的途径,植物内生固氮菌资源的挖掘和利用对我国农业可持续发展具有重要实践意义。【目的】筛选高效甘蔗内生固氮菌,并对其联合固氮效率及促生长功能进行评价。【方法】从广西甘蔗茎基部组织分离筛选到一株内生固氮菌株NN08200,利用乙炔还原法测定固氮酶活性,通过菌落PCR扩增nif H基因确定菌株为固氮菌;通过菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析确定该菌株的分类;采用盆栽接种测定菌株对甘蔗的实际促生长作用,并利用15N同位素稀释法测定其相对固氮效率。【结果】菌株NN08200的固氮酶活性达到2445nmolC2H4/(h·m L),菌株的nif H基因长度为339bp,与甘蔗内生固氮醋酸杆菌Gluconacetobacter diazotrophicus PAL5菌株的nif H相似性达99%;根据菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析结果,菌株NN08200属于泛菌属(Pantoeasp.)细菌;盆栽接种菌株NN08200能显著提高甘蔗幼苗的株高和干重,15N同位素分析结果表明接种该菌株甘蔗植株的根、茎和叶从空气中获得氮素的百分率分别为7.49%、15.02%和10.79%,其联合固氮效率显著优于甘蔗内生固氮模式菌株G. diazotrophicus PAL5,利用后者接种的甘蔗根、茎和叶从空气中获得氮的百分率分别为3.53%、9.44%和4.87%。【结论】菌株Pantoea sp. NN08200是高效甘蔗内生固氮菌,其固氮促生长效果明显高于G. diazotrophicus PAL5菌株,可望研发成为优良固氮微生物肥料生产菌种,并可进一步用于甘蔗联合固氮菌作用机理的相关研究。     

In vitro strategies for selection of eye-spot resistant sugarcane lines using toxins of Helminthosporium sacchari

In vitro strategies were applied for the selection of eye-spot resistant variants from susceptible sugarcane cultivar Co 419 Different selective units (callus and leaf) of the susceptible cultivar were subjected to sub-lethal to lethal doses of toxins (culture filtrate and partially purified toxin) of H. sacchari, with the objective of improving the efficacy of in vitro selection protocols. All the selective units gave more or less similar response with culture filtrate, but a distinct response was observed when leaf was subjected to partially purified toxin treatment. The response was characterised by the degree of resistance exhibited by the regenerated seedlings.     

N-fertilizer saving by the inoculation of Gluconacetobacter diazotrophicus and Herbaspirillum sp. in micropropagated sugarcane plants

Colonization of micropropagated sugarcane plants by Gluconacetobacter diazotrophicus and Herbaspirillum sp. was confirmed by a dot-immunoblot assay. In all, a 45-day short-term and 180-day long-term experiments conducted on micropropagated sugarcane plants of Co 86032, a sugar rich popular variety in South India, indicated the usefulness of these diazotrophs as plant growth promoting bacteria. Co-inoculation of these two bacteria enhanced the biomass considerably under N-limited condition in the short duration experiment. In the long-term experiment, the establishment of inoculated Herbaspirillum sp. remained stable with the age of the crop up to 180 days, while there was a reduction in population of G. diazotrophicus for the same period. The total bio-mass and leaf N were higher in plants inoculated with G. diazotrophicus and Herbaspirillum sp. without N fertilization and also in plants with 50% of the recommended N (140 kg ha(-1)) than the plants fertilized with recommended dose of inorganic N (280 kg ha(-1)). This experiment showed that inoculation with these bacteria in sugarcane variety Co 86032 could mitigate fertilizer N application considerably in sugarcane cultivation.     

Isolation and identification of Gluconacetobacter azotocaptans from corn rhizosphere

Six acetic acid producing, diazotrophic bacteria were isolated from soil adhering to corn roots. These isolates were shown to be Gluconacetobacter azotocaptans and they shared some features with G. johannae and G. diazotrophicus but differed on the basis of colony morphology on different media, use of carbon sources and use of l-amino acids as a nitrogen source. The species identity was confirmed using 16S rDNA sequence analysis, PCR amplification of 16S rRNA gene with species-specific primers and amplified rDNA restriction analysis. This is the first report of the presence of this bacteria on corn plants. Scope of the paper: This is the first report of the occurrence and association of Gluconacetobacter azotocaptans with corn.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Vector transmission of Banana streak virus in the screenhouse in Uganda

Although mealybug transmission of Banana streak virus.(BSV) by Planococcus citri and Saccharicoccus sacchar has been demonstrated elsewhere, these mealybugs have not been identified on bananas in Uganda and their role and that of other agents in BSV transmission is not well documented. Insect samples were collected from banana farms in sites with low, moderate and high BSV infections in Uganda. Subsequently, live mealybugs and aphids were again collected and used in acquisition, retention and transmission tests, and BSV diagnosed using TAS‐ELISA. Dysmicoccus brevipes (pineapple mealybug), S. sacchari (sugarcane mealybug) and Pentalonia nigronervosa (banana aphid) were the most abundant insect species from banana fields sampled. Abundance of D. brevipes was positively and significantly correlated with BSV incidence unlike that of. P. nigronervosa. Transmission studies in the screenhouse showed that mealybugs acquired BSV one day after feeding on virus sources and approached optimum acquisition after the third day. Pineapple and sugarcane mealybugs retained BSV up to 5 days from the day of transfer from the virus source. BSV was first detected in the recipient banana plants 4 wk after transmission using pineapple mealybug and 6 wk after inoculation using sugarcane mealybug. Under screenhouse conditions, both mealybugs therefore appear to transmit BSV semipersistently.     

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

Detection of sugarcane yellow leaf virus,the causal agent of yellow leaf syndrome in sugarcane by DAS-ELISA

DAS-ELISA studies were conducted on detection of sugarcane yellow leaf virus (SCYLV) causing yellow leaf syndrome (YLS) of sugarcane in leaf and juice antigens. Among the two types of antigen sources used for the virus detection, juice antigen showed high titre for the virus as compared to leaf antigen. Assay with juice samples recorded more number of varieties positive to the virus. Further DAS-ELISA studies revealed that plants raised from disease-infected planting materials recorded high titre for SCYLV as compared to those raised from symptom-free seed canes. Similarly, assaying SCYLV titre in plant and ratoon crop in the field showed that SCYLV infection was partial in plant crop and in the subsequent ratoon crop, all the samples were positive to the virus. ELISA studies also indicated that 33 of 41 cane varieties showing YLS were positive to the virus.     

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.     

Impact of Gyranusoidea tebygi Noyes (Hymenoptera: Encyrtidae) on the Mango Mealybug Rastrococcus invadens Williams (Homoptera: Pseudococcidae) in Nigeria

This study investigated the impact of released exotic mango mealybug parasitoid Gyranusoidea tebygi on mango mealybug Rastrococcus invadens in Nigeria. Observations were also made on the occurrence of the mealybug on other host plants in the surveyed areas. The monitoring exercise started in 1991 about 2 years after the first release in Ibadan. By 1997 and 1998, G. tebygi was found to have crossed all agro-ecological barriers to colonize the entire area of infestation nationwide on mango as well as other host plants. During this period, the populations of R. invadens had greatly decreased from between 11.0 and 98.0 mealybugs per leaf in 1991 to between 0.0 and 18.2 mealybugs per leaf in 1998. This fall was attributed to the activities of the released parasitoid. At many sampling sites in 1998, mealybugs were virtually absent on both mango and other host plants. Predators that were observed during the survey, were the coccinellids: Exochomus promptus Weise, Chilocorus nigritus (F) and Nephus spp. Larvae of chrysopid species, Ceratochrysa autica (Walker) and Plesiochrysa sp. The hyperparasitoids reared from mummies were Marietta leopardina Motsch (Aphelinidae), Chartocerus hyalipennis Hayat and Chartocerus subaeneus (Forster) (Signiphoridea).     

Quantitative proteomic analysis of the interaction between the endophytic plant-growth-promoting bacterium Gluconacetobacter diazotrophicus and sugarcane

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium that colonizes sugarcane. In order to investigate molecular aspects of the G. diazotrophicus-sugarcane interaction, we performed a quantitative mass spectrometry-based proteomic analysis by (15)N metabolic labeling of bacteria, root samples, and co-cultures. Overall, more than 400 proteins were analyzed and 78 were differentially expressed between the plant-bacterium interaction model and control cultures. A comparative analysis of the G. diazotrophicus in interaction with two distinct genotypes of sugarcane, SP70-1143 and Chunee, revealed proteins with fundamental roles in cellular recognition. G. diazotrophicus presented proteins involved in adaptation to atypical conditions and signaling systems during the interaction with both genotypes. However, SP70-1143 and Chunee, sugarcane genotypes with high and low contribution of biological nitrogen fixation, showed divergent responses in contact with G. diazotrophicus. The SP70-1143 genotype overexpressed proteins from signaling cascades and one from a lipid metabolism pathway, whereas Chunee differentially synthesized proteins involved in chromatin remodeling and protein degradation pathways. In addition, we have identified 30 bacterial proteins in the roots of the plant samples; from those, nine were specifically induced by plant signals. This is the first quantitative proteomic analysis of a bacterium-plant interaction, which generated insights into early signaling of the G. diazotrophicus-sugarcane interaction.     

Binomial sequential sampling of adult Saccharicoccus sacchari on sugarcane

The between-stalk dispersion characteristics of adults of the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) were determined in southern Queensland. Iwao's patchiness regression was inappropriate to describe the relationship between mean and variance. Taylor's power law indicated that adults were aggregated, especially at the beginning and end of the ratoon growth period. Binomial data were modelled by the Nachman model; the model of Grout and two models of Wilson & Room were inappropriate to describe the relationship between proportion of stalks infested and mean numbers of adults per stalk. Relationships to determine sample sizes for fixed levels of precision and binomial fixed-precision-level stop lines are developed for different sampling times using Taylor's power law and Nachman's equation.     

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene codes for 130-kDa toxin protein

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.     

甘蔗天冬氨酰半醛脱氢酶基因的电子克隆与分析

应用电子克隆技术,以水稻EF576477序列为探针,获得了甘蔗天冬氨酰半醛脱氢酶基因（aspartate．semialdehydedehy—drogenase,ASADH）的一条cDNA全长序列,命名为ScASADH。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、理化性质、亚细胞定位、跨膜结构域、疏水性／亲水性、高级结构及功能域等方面进行预测和分析。结果表明：该基因全长1711bp,包含一个1128bp的开放阅读框,编码375个氨基酸,该基因编码蛋白定位于细胞核,为可溶性蛋白,存在信号肽,二级结构原件多为无规卷曲,含有多个保守功能域,主要功能为翻译。电子表达分析结果显示,该基因在甘蔗根尖、幼苗、花序、叶片和茎中组成型表达,其中在茎中的表达量比其他组织类型中表达量高。该基因的表达受葡萄糖杆菌和赤腐病菌的调控。     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.