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.     

Numbers of pink sugarcane mealy bug, Saccharicoccus sacchari (Cockerell) (Hemiptera: Pseudococcidae), differ within seasons and among regions and stages of the sugarcane crop cycle

Abstract  The pink sugarcane mealy bug (PSMB; Saccharicoccus sacchari ) is widespread on sugarcane globally. PSMB infest above-ground storage tissue as it develops, feeding on phloem and producing exudate. It is not known, however, whether the level of infestations is the same in different sugar growing regions, or how population size varies year to year within a region. Field surveys of the number of nodes infested were conducted over five seasons in three mill-regions in northern Australia (Macknade, Kalamia and Marian) on plant and ratoon crops. The pattern of infestation was very similar across seasons (only in 1 year of very low rainfall was the increase in population delayed). In all three regions the proportion of nodes infested was similar but reached the maximum 1 month later in the Marian region compared with the Kalamia and Macknade regions. The Kalamia region was distinguished by the rapid decline in the number of nodes infested down to a very low level by March. In the Macknade region mealy bugs persisted at higher levels than the other two regions. The PSMB infestation started earlier and was much greater in ratoon crops than plant crops throughout the sampling period. The differences were more pronounced in the Macknade and Marian districts. These observations provide a firm basis from which future strategies to control PSMB can be developed.     

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.     

Field and laboratory studies on the fungus Aspergillus parasiticus, a pathogen of the pink sugar cane mealybug Saccharicoccus sacchari

Infestation of sugar cane nodes by the mealybug Saccharicoccus sacchari (Cockerell) was studied in two commercial fields over a 7-month period in 1987. Natural enemies associated with S. sacchari were fungi Aspergillus parasiticus Speare, Metarhizium anisopliae (Metschnikoff) Sorokin, and Penicillium spp.; the dipteran Cacoxenus perspicax Knab; and the hymenopteran parasitoid Anagyrus saccharicola Timberlake. A. parasiticus was the predominent natural enemy of S. sacchari whereas all other natural enemies showed a low level of activity. The highest prevalence of A. parasiticus was in March when it occurred on 84% of S. sacchari-infested nodes. The prevalence of A. parasiticus declined rapidly during April and May and was absent in the winter months during which nodal infestation of S. sacchari increased. In laboratory bioassays all fungal isolates originating from S. sacchari were more virulent at 28°C than at 24°C. Laboratory studies supported the hypothesis based on field observations that temperature highly influenced the efficacy of A. parasiticus against S. sacchari.     

Soil sampling for <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> spores in Queensland sugarcane fields

Three sugarcane fields in Bundaberg and four fields in each of the Burdekin, Tully and Innisfail (Queensland, Australia) were sampled for spores of Metarhizium anisopliae (Metchnikoff) (Deuteromycotina: Hyphomycetes). This entomopathogenic fungus is the active ingredient in the biocide “BioCane^TM”, which was developed for the management of the greyback canegrub Dermolepida albohirtum (Waterhosue) (Coleoptera: Scarabaeidae) and other scarabs in cane fields. Fields sampled were of different crop ages and all had a history of BioCane^TM treatment in Plant Cane in past years. Soil samples were taken in each field from four depths (0–10, 10–20, 20–30 and 30–40 cm below soil level) with the use of an auger. Spore levels were highest at the depths of 10–20 and 20–30 cm. Spore levels differed between locations with Innisfail and Tully recording the highest spore counts. Spores were also found in the inter-row space in plots sampled in Tully. Sampling statistics were determined for M. anisopliae spores at the four soil depths with 0.1 and 0.25 precision levels. Three sampling methods were compared (use of marker beads; use of 100 mm auger and 150 mm auger). Samples that relied on marker beads resulted in higher spore counts, however, an auger can still be used since BioCane^TM does not normally contain coloured markers. Results obtained demonstrate the ability of the pathogen to translocate in soil profile and across rows, most likely due to grub movements and other soil fauna. Sampling for M. anisopliae spores provides good monitoring of their levels in soil. The implications of this on grub management decisions are discussed.     

Photographic sampling: a photographic sampling method for mites on plants

A photographic sampling method for mites on plants was evaluated using Tetranychus urticae and Phytoseiulus persimilis on pepper plants. It was found to be 92% accurate for T. urticae eggs and 98% accurate for P. persimilis eggs at densities up to 45 eggs per cm² for T. urticae, and up to 3 eggs per cm² for P. persimilis. The motiles of the two species were not confused, nor were they confused with exuviae or other matter.     

Taxonomy ofPuccinia species causing rust diseases on sugarcane

A taxonomic revision ofPuccinia species causing rust diseases on sugarcane was conducted to clarify their morphological characteristics. Specimens including previously reported species,Puccinia melanocephala, P. kuehnii andPuccinia sp.sensu Muta, 1987, were collected in Japan and the Philippines and borrowed from various herbaria worldwide. Morphological characteristics of these specimens were examined under light and scanning electron microscopes. Comparative morphological studies of the specimens showed that rust fungi infecting sugarcane could be classified into two species,Puccinia melanocephala andP. kuehnii. Puccinia sp.sensu Muta was morphologically identical withP. kuehnii. Results of this study corroborate previous phylogenetic analysis results of D1/D2 regions of LSU rDNA gene. Contribution No. 157, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Efficiency of variable-intensity and sequential sampling for insect control decisions in cole crops in the Netherlands

A total of 24 commercial fields of cabbages and Brussels sprouts were sampled in a grid fashion with 20–25 equally spaced cells with four plants per cell. Using this data base of 80–100 plants, we conducted computer stimulations to compare the treatment decisions that would be made for the major insect pests using published sequential sampling programs and a newly developed variable-intensity sampling program. Additionally, we compared the number of samples required to make the decision. At low thresholds (10–20%) for both Lepidoptera and cabbage aphids, variable intensity-sampling required a smaller sample size and provided more reliable decisions, while at high thresholds (40–50%) sequential sampling provided more reliable decisions. In both procedures, the occurrence of incorrect decisions was minimal. The number of cases in which a decision would not be reached after a 40-plant sample was lower for variable-intensity sampling. Considering the number of samples required to make a correct decision and the greater need for reliable decisions at lower thresholds, variable-intensity sampling was superior to sequential sampling. Additionally, variable-intensity sampling has the advantage of requiring samples to be taken in a greater area of the field and thus increases the probability of detecting localized infestations. Although variable-intensity sampling was not designed to classify pest populations for treatment decisions but rather to achieve sampling precision around the population mean, our present studies indicate that it can also be an effective method to aid in treatment decisions.     

Spatial distribution and sequential sampling of adults ofGonocephalum macleayi andPterohelaeus darlingensis in different cropping regimes

Counts of adults of the false wirewormsGonocephalum macleayi (Blackburn) andPterohelaeus darlingensis Carter in pitfall traps in burnt, mulched and sorghum treatments conformed to Taylor's power law. Within a species there were no significant differences in distributions of counts of either sex in any habitat butG. macleayi were more aggregated thanP. darlingensis (Taylor'sb 1.35 and 1.26, respectively). Relationships to determine sample sizes for fixed levels of precision and fixed-precision-level stop lines for sequential sampling are developed for each species using Taylor's parameters for combined data over all habitats.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

甘蔗鞭黑粉菌侵染甘蔗的早期可视化观察

[目的]甘蔗鞭黑粉菌(Sporisorium scitamineum)引起的甘蔗黑穗病是我国甘蔗生产重要的病害.示踪甘蔗鞭黑粉菌侵染甘蔗的过程将有助于揭示其致病性和甘蔗抗黑穗病机制,为抗病品种的选育以及黑穗病的防治奠定基础.[方法]利用农杆菌介导的遗传转化技术对甘蔗鞭黑粉菌进行黄色荧光标记,对转化子进行配合及致病力检测...     

甘蔗ScNRAMP基因家族的鉴定与生物信息学分析

NRAMP蛋白(natural resistance-associated macrophage proteins)家族在植物响应重金属胁迫时具有重要作用,能够转运Fe²⁺、Mn²⁺、Zn²⁺和Cd²⁺等重金属离子。为探究甘蔗ScNRAMP基因家族的特征,该文基于甘蔗割手密基因组鉴定了ScNRAMP基因家族,并进行了理化特性、基因结构、顺式作用元件、保守基序、结构域和进化关系等分析。结果表明:甘蔗ScNRAMP基因家族含有29个成员,不均匀分布在19条染色体上; 编码蛋白均为不稳定蛋白,无信号肽,亚细胞均定位在质膜上; 各成员保守基序有6～10个不等,跨膜数有6～12个不等,二级结构主要构成元件为α-螺旋和无规则卷曲; 顺式作用元件分析表明甘蔗ScNRAMP基因家族可能通过激素代谢参与逆境胁迫和生长发育等生物过程; 利用割手密的RNA-seq转录组表达数据进行的组织特异性分析发现,ScNRAMP在甘蔗不同发育阶段的叶和茎中具有时空表达特性; 进化树分析将甘蔗ScNRAMP家族成员分为3个亚家族(I、Ⅱ和Ⅲ)。该研究在全基因组水平上系统地鉴定了现代栽培甘蔗祖先种之一割手密NRAMP基因家族,既为进一步了解甘蔗NRAMP基因家族提供了基础,也为后续甘蔗重金属研究提供了重要候选基因。     

Experimental evaluation of the efficiency of Epidinocarsis lopezi, a parasitoid introduced into Africa against the cassava mealybug Phenacoccus manihoti

The capability of Epidinocarsis lopezi (De Santis) (Hymenoptera: Encyrtidae) to control the cassava mealybug (CM) Phenacoccus manihoti Mat.-Ferr. (Homoptera: Pseudococcidae) was investigated in Nigeria using physical and chemical exclusion experiments. In two sleeve cage experiments CM populations, about 2 months after artificial infestation, were 7.0 and 2.3 x lower on artificially infested cassava tips covered with open cages than on tips in closed cages which excluded most parasitoids. On similarly infested but uncovered tips, CM populations were 24.3 and 37.5 x lower, and parasitisation rates were higher. In an artificially infested field which was treated weekly with carbaryl, parasitisation rates were below 10% and CM populations exceeded 200 per tip. In the chemically untreated plot, parasitisation rates were up to 25% and CM densities were mostly below 10 per tip. This study demonstrates the efficiency of E. lopezi in controlling its host under the experimental conditions.

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Résumé La capacité d'E. lopezi de contrôler la cochenille farineuse du manioc a été évaluée au Nigéria en excluant le parasitoïde de son hôte par des moyens physiques et chimiques. Dans deux expériences utilisants des manchons les populations de la cochenille deux mois après l'infestation artificielle étaient 7.0 et 2.3 fois plus basses sur les branches couvertes d'un manchon ouvert que sur les branches couvertes par un manchon fermé, qui excluait la plupart des parasitoïdes. Sur les apex sans manchons, également infestés artificiellement, les populations de la cochenille étaient 24.3 et 37.5 fois plus bas tandis que le degré de parasitisme était plus élevé. Dans un champ infesté artificiellement et partiellement traité chaque semaine avec du carbaryl, le pourcentage de parasitisme restait au-dessous de 10%, et les populations de la cochenille dépassaient 200 par apex. Dans la partie non-traitée, le parasitisme atteignait 25% et la densité de la cochenille restait pour la plupart du temps au-dessous de 10 cochenilles par apex. Ces expériences démontrent la capacité d'E. lopezi de maintenir son hôte à un bas niveau dans des conditions expérimentales.

     

Laboratory and field evaluation ofAllorhogas pyralophagus for the control of lepidopterous borers infesting sugarcane in tropical India

All the seven species of lepidopteran insects viz.,Chilo infuscatellus Snell.,C. sacchariphagus indicus (Kapur),C. partellus Swinhoe,Scirpophaga excerptalis Walker,Sesamia inferens Walker,Corcyra cephalonica Staint., andGalleria mellonella (L.) were parasitized under laboratory conditions by the imported braconid parasite,Allorhogas pyralophagus Marsh. Among theseS. excerptalis was the most suitable host for the multiplication of the parasite. When commonly occurring borer pests of sugarcane in tropical India were offered in single and multiple choice test, the parasite preferredS. excerptalis compared toc. infuscatellus andC. sacchariphagus indicus. During 1988 and 1989 growing seasons, attempts were made to colonizeA. pyralophagus againstC. sacchariphagus indicus andS. excerptalis attacking sugarcane. A total of 11,754 and 15,651 mated females were released in 0.4 ha sugarcane field during 1988 and 1989 respectively, but the parasite failed to colonise on the sugarcane borers.      

Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets

Calli have been initiated from young leaves of in vitro grown sugarcane shoots. Histological examination has shown that the two types of calli induced (nodular and friable) originated from different regions of the explants and were cytologically different.This study has shown an obvious relation between the developmental stage of the excised tissue and the potential of plant regeneration of the in vitro initiated callus culture. Nodular calli were obtained from bases of the fast-growing young leaves while their more mature parts of the older leaves only produced friable calli. High-frequency plant regeneration via somatic embryogenesis was obtained from nodular calli while friable calli rarely produced plantlets.     

Ribosomal DNA-ITS sequence polymorphism in the sugarcane rust,Puccinia kuehnii

The two highly divergent ITS types previously observed in isolates ofPuccinia kuehnii were amplified from the same isolates through the use of ITS type-specific primers for PCR and are therefore considered as polymorphisms of ITS regions within the species. Although homology of sequences of one of the ITS types with otherPuccinia species was of expected levels, significantly high homology of sequences of the other type with those ofCronartium members indicates that abnormal genetic events led to the occurrence of these polymorphic regions. These results indicate that ITS gene tree phylogeny may not reflect true species phylogeny in this group of rust fungi. Rather, D1/D2 region tree phylogeny, which was concordant with the differences in morphology with and among related rusts, more correctly reflects phylogenetic relationships of sugarcane and related grass rusts. contribution No. 159, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.