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.     

Effects of biocides and rotation breaks on soil organisms associated with the poor early growth of sugarcane in continuous monoculture

Glasshouse and field experiments were conducted to determine the effects of biocides and rotation breaks on deleterious soil organisms associated with the poor early growth and subsequent yield decline of sugarcane grown in continuous monoculture. Fumigation of a soil that had been under sugarcane monoculture with minimal breaks for more than 30 years markedly improved the health and growth of the sugarcane sett and shoot root systems, increased the growth of the primary shoot and stimulated more and larger secondary shoots. It also reduced populations of culturable fungi in the rhizosphere of the sett roots and reduced colonization of the sett and shoot roots by lesion nematode (Pratylenchus zeae). Exposure of the developing sett root system for 14 days to mono-cultured sugarcane soil was sufficient to significantly retard subsequent plant growth. In field experiments, fungicide and nematicide (mancozeb + aldicarb), when applied together to land under sugarcane monoculture, was as effective as fumigation in improving early sugarcane growth and increasing sugarcane yields. Rotation breaks (alternate crops, sown pasture, bare fallow) that were in place for 54 months, increased sugarcane establishment and increased sugarcane yields to levels similar to that obtained following fumigation of land under sugarcane monoculture. Fumigation of land that had been under the rotation breaks gave plant growth responses that were in addition to that achieved by the breaks alone. A mancozeb + aldicarb treatment was as effective as fumigation in increasing sugarcane yields after a bare fallow break but accounted for only a portion of the fumigation response following the crop and pasture breaks. Improved plant nutrition may be a factor in the fumigation response following the crop and pasture breaks. Plant growth responses to fumigation and the manocozeb + aldicarb treatments that were manifested in final sugarcane yields (after one years growth) were evident as plant growth responses (sett root, shoot root and primary shoot dry weight) measured 54 days after planting. The experimental results support the concept that when sugarcane is grown as a monoculture, deleterious fungi and nematodes retard plant establishment and early plant growth and that this leads to reduced sugarcane yields.     

Distribution and sequence analysis of the centromere-associated repetitive element CEN38 of Sorghum bicolor (Poaceae)

Fluorescence in situ hybridization (FISH) of a large-insert genomic clone, BAC 22B2, previously suggested that Sorghum bicolor (2n = 20) has the tetraploid architecture A(b)A(b)B(b)B(b). Here, we report on BAC 22B2 subclone pCEN38 (1047-bp insert) as related to sorghum and sugarcane. Mitotic FISH of six different subclones of BAC 22B2 showed that pCEN38 produced the strongest specificity to the A(b) subgenome and signal occurred primarily near centromeres. Southern blots of pCEN38 to 21 crop plants revealed a narrow taxonomic distribution. Meiotic metaphase I FISH positioned pCEN38 sequences near active centromeres. Pachytene FISH revealed that the distributions are trimodal in several B(b) and possibly all sorghum chromosomes. DNA sequencing revealed that the pCEN38 fragment contains three tandemly repeated dimers (<280 bp) of the same sequence family found in sorghum clone pSau3A10, and that each dimer consists of two divergent monomers (<140 bp). Sequence comparisons revealed homology between the pCEN38 monomers and the SCEN 140 bp tandem repeat family of sugarcane. FISH of pCEN38 yielded signal in centromere regions of most but not all sugarcane chromosomes. Results suggest that sugarcane and sorghum share at least one ancestor harboring elements similar to pCEN38 and SCEN and that each species had an ancestor in which the repetitive element was weakly present or lacking.     

Effect of varying co2 and light levels on growth of hedyotis and sugarcane shoot cultures

Summary Shoot cultures of Hedyotis corymbosa, a C3 species, and sugarcane, a C4 species, were used to examine the effects of various CO₂ concentrations and two light intensities on growth and photosynthetic rates. The fresh and dry weights of new growth of Hedyotis shoots were higher when grown under the higher light intensity, while differences among shoots grown under different CO₂ levels were marginal. After 14 d of growth in various CO₂ concentrations, no significant differences could be observed in the newly produced leaves of Hedyotis with respect to stomatal distribution and number of mesophyll cell layers. Shoots grown under high light intensity did not show higher rates of photosynthesis than those grown under low light intensity. Also, sugarcane shoots grown in a CO₂-enriched environment did not have higher photosynthetic rates, perhaps because the C4 pathway is less sensitive to the ambient CO₂ concentration. The quantum yield of Hedyotis shoots grown on medium with 20 g l⁻¹ sucrose was lower than that of shoots on lower sucrose concentrations, supporting the view that photosynthesis is inhibited by high levels of sucrose. Our results suggest that Hedyotis shoots in culture exhibit some form of acclimation to high CO₂. so that there is no net gain in productivity by photosynthesis.     

新垦红壤坡地土壤水分有效性研究

针对南方红壤地区降雨时空分布不均的特点,以未开垦的自然植被为对照,对桂西北环境移民示范区不同季节(早季和雨季)一次性降雨前1d及降雨后4h、2d、4d、6d及8d新垦蔗地(中坡、下坡、谷地）0-20cm,20-40cm、40-60cm3个土层的土壤水分含量进行了测定．结果表明,雨后谷地蔗地的土壤有效水分增量几乎与降雨量相同,而中坡蔗地与未开垦的自然植被土壤有效水分增量仅相当于降雨量于80％．雨季雨后0-60cm土壤层次中土壤有效水分分布均匀,早季主要集中在表层．雨季一次性降雨后各哩理及各土层土壤有效水分饱和度均有显著差异;而早季3个新垦蔗地间无明显差异,3个土层以表层土壤有效水分饱和度最高,亚表层与心土层差异不明显．无论雨季还是旱季,0-60cm土层土壤有效水分的消耗速率都以自然植被处理为最低,由于雨季正是作物生长旺季,其0-60cm土层土壤有效水分的消耗速率比旱季快,按照早季雨后8d土壤有效水分的平均消耗速率,15d内0-60cm土层的有效水分将消耗殆尽。     

Functional response of Trichogramma chilonis to Galleria mellonella and Chilo sacchariphagus eggs

A biological control programme using inundative releases of Trichogramma chilonis Ischii (Hymenoptera: Trichogrammatidae) reared on Galleria mellonella L. (Lepidoptera: Pyralidae) is currently underway to reduce infestations of Chilo sacchariphagus Bojer (Lepidoptera: Crambidae) in sugarcane, Saccharum spp., on Réunion Island. To assess the potential of the parasitoid as an inundative biocontrol agent, the functional response of three T. chilonis strains was tested with G. mellonella and one strain with C. sacchariphagus host eggs in glass tubes in the laboratory. The shape of the functional response (type II or III) was determined using logistic regression, and attack coefficients and handling times (T_h) were determined using non‐linear least‐square regression. The behaviour of all three strains with G. mellonella host eggs corresponded to a type III response. The St Benoît T. chilonis strain had a significantly shorter estimate of T_h than the St Pierre strain (P<0.05) and may, therefore, be more appropriate as a biocontrol agent. The functional response with C. sacchariphagus host eggs was a type II with the St Benoît T. chilonis strain. More T. chilonis wasps developed per host egg from the larger C. sacchariphagus host eggs (2.9) relative to G. mellonella (1.1). Superparasitism at low host egg densities was, therefore, likely to have been less frequent with C. sacchariphagus. Black eggs were chosen as an estimate of number of eggs parasitized, although they represent the number of eggs where parasitism led to complete pupal development. The low rate of detected parasitism at low host densities with G. mellonella eggs may be due to incomplete pupal development due to superparasitism rather than lack of parasitism, thus explaining the type III functional response.     

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C₄ plants for the production of poly[(R)‐3‐hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield‐potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β‐ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl‐CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB‐producing sugarcane containing the β‐ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10⁶ Da. These results are a major step forward in engineering a high biomass C₄ grass for the commercial production of PHB.     

锰胁迫对甘蔗幼苗缺铁和失绿的影响

近年来,甘蔗主产区的甘蔗幼苗出现严重的缺铁失绿问题,影响了我国甘蔗生产及食糖安全。为了揭示锰诱导甘蔗幼苗缺铁失绿机制,该研究采用水培试验法,对过多锰诱导的甘蔗幼叶失绿及其与铁素营养的关系进行了探讨。结果表明:过多锰胁迫下随着甘蔗中锰含量的增加,幼叶明显失绿。250、500、750μmol·L-1处理10 d后,幼叶中的叶绿素含量分别从对照处理的1.71 mg·g-1FW下降至0.86、0.85、0.64 mg·g-1FW。过多锰抑制甘蔗对铁的吸收,每株植株对铁吸收量(3.22~4.40 mg)显著减少。幼叶中铁含量(116.8~128.6 mg·kg-1DW)也随着锰处理浓度的增加而显著降低。250~750μmol·L-1处理的甘蔗幼叶中铁的含量仅相当于对照处理的89.4%~81.2%。相反,锰处理后根和茎中铁的含量却显著增加。锰胁迫下幼叶中活性铁含量和活性铁与全铁比值(0.14~0.21)也显著降低。高锰胁迫下,幼叶中的活性铁含量(4.1~6.9 mg·kg-1FW)相当于对照处理的25.5%~55.2%。相关分析结果显示,锰胁迫下的甘蔗叶片中活性铁含量与叶绿素含量呈显著的正相关;锰处理后幼叶中活性铁与锰含量的比值从对照的0.71下降至0.04~0.01。这说明过多的锰可诱导甘蔗幼叶失绿,而失绿与过多的锰胁迫下甘蔗对铁的吸收、运输受阻及铁的钝化有关。     

Virulence of Beauveria brongniartii and B. bassiana against Schizonycha affinis white grubs and adults (Coleoptera: Scarabaeidae)

Two endemic scarab pests, Schizonycha affinis Boheman and Hypopholis sommeri Burmeister (Coleoptera: Melolonthinae) have increased in prevalence in the sugarcane producing regions of the KwaZulu‐Natal Midlands, South Africa. The crop losses associated with their feeding, the failure of chemical insecticides applied for their control, and the recent discovery of Beauveria brongniartii (Sacc.) Petch (Ascomycota: Cordycipitaceae) epizootics on these pests, have generated interest in the development of a mycoinsecticide targeting adults and larvae of these species. Previous research, using microsatellite markers, identified low levels of genetic diversity among isolates of B. brongniartii collected from two field sites where epizootics occurred. The virulence of 21 of these closely related B. brongniartii isolates and two isolates of Beauveria bassiana (Balsamo) Vuillemin was evaluated. Bioassays were conducted against adults and larvae of S. affinis, and adult Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) as a surrogate test insect. The closely related B. brongniartii isolates varied significantly in their virulence towards both S. affinis (50.1–95% mortality) and T. molitor (39–74% mortality), with a number of these not highly virulent against either of these insect species. Those isolates sharing a haplotype did not vary in virulence. Adults of S. affinis were more susceptible than larvae to isolates of B. brongniartii. The median lethal concentration (LC₅₀) required to kill half the adult S. affinis test insects was 7.65 × 10⁶ conidia per millilitre. Schizonycha affinis second instar larvae had a median survival time of 17.5 days when exposed to some B. brongniartii isolates; however, third instars survived significantly longer with a median of 21 days. Third instars exposed to the highest concentration of B. brongniartii isolate HHWG1 survived for a median time of 15 days. Bioassays supported the finding that genetically closely related isolates may vary in their virulence, even if they were obtained from the same field epizootics.