A Method for Selection of Tolerance to African Cassava Mosaic Virus

Detopping hastened the appearance of mosaic symptoms in shoots of cassava cuttings infected with African cassava mosaic virus (ACMV). The degree of plant recovery from severe mosaic after each detopping was the basis for evaluation of ACMV tolerance. In field-screening, detopping of cassava shoots infected with ACMV is recommended for selection of tolerance to the virus. One detopping of ACMV-infected shoots was sufficient to analyze tolerance level reliably. Out of 521 lines only one was found tobe tolerant, while 144 lines were found to be moderately tolerant. These included the lines ‘Gimbi MA 235’, (02945S × 3119)S MA 219, and ‘Kadanga Malombe’ previously described as resistant to ACMV.     

Incidence of Three Major Cassava Diseases on Local Susceptible Cassava Cultivars at Three Planting Dates

Incidence of African cassava mosaic (ACMD), cassava bacterial blight (CBB), and cassava anthracnose (CA) on local susceptible cassava cultivars planted in December, February, and April was investigated. December cassava planting had a higher incidence of ACMD, CBB, and CA diseases compared with February and April plantings. CA seemed to be more prevalent at an older stage (8 months old) of plant growth. April planting had significantly lower incidence of major cassava diseases, and higher mean tuber, yield than the same cassava cultivars planted earlier. The length of exposure of cassava plants to the inocula and/or to the vector during the rainy season determines the occurrence of diseases. In the absence of resistant cassava cultivars and through, partial disease escape of susceptible cultivars from ACMD, CBB, and CA, planting the same cultivars in April can still produce a profitable cassava crop.     

Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina

Pathogenesis-related (PR) proteins are induced in response to pathogen attack. In the present study, the induction of PR proteins in response to the fungal pathogen Macrophomina phaseolina was investigated in 15-day- and 1-month-old plants of Vigna aconitifolia with resistant and susceptible cultivars. Inoculation of the fungal pathogen resulted in the enzyme activity gradually increased throughout the experimental period of 168 h compared to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in the resistant FMM-96 cultivar as compared to susceptible RM0-40 and CZM-3 cultivars. Furthermore, the western blot analysis revealed the presence of 33- and 30-kDa bands of β-1,3-glucanase and chitinase in induced moth bean plants, respectively. The possible implications of these findings as part of the general defense response of moth bean plants against the fungal pathogen (M. phaseolina) have been discussed.     

QTL analysis of field resistance to Xanthomonas axonopodis pv. manihotis in cassava

We evaluated cassava bacterial blight (CBB) infection in an pair-cross population of 150 individuals derived from an intra-specific cross between two non-inbred cassava (Manihot esculenta Crantz) lines. The replicated trials were carried out in the field under high disease pressure over two consecutive crop cycles. Evaluations were conducted at 4 and 7 months after planting for the two cycles. Simple regression analysis and the nonparametric Kruskal-Wallis rank-sum test revealed that eight quantitative trait loci (QTLs) were involved in resistance. We detected changes in QTLs from crop cycle to crop cycle. The pathogen population (Xanthomonas axonopodis pv. manihotis) was also monitored over the period, using a restriction fragment length polymorphism probe and pathogenic tests. Changes in QTL detection over the 2 years could be correlated with changes in pathogen population structure. One QTL, located in linkage group D, was conserved over the two crop cycles, and in field to greenhouse evaluations. This study thus identified molecular markers useful for marker assisted-selection, a technique that can accelerate the long, multiple-season process of breeding for CBB resistance. Received: 1 January 2000 / Accepted: 25 June 2000     

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Cassava Bacterial Blight: Using Genomics for the Elucidation and Management of an Old Problem

Bacterial Blight is an important disease of cassava, causing losses that have resulted in historical famines in certain growing zones. The disease is caused by Xanthomonas axonopodis pv. manihotis, a gram-negative rod that belongs to the gammaproteobacteria. In this review, we describe the pathosystem and the recent studies that have been undertaken to elucidate both susceptibility and resistance mechanisms in cassava, with the hope of generating resistant plants using biotechnology. We first describe studies of the pathogen, including pathogen population changes through time as well as genomic tools that have recently been generated to determine pathogenicity factors. Secondly, we discuss mechanisms of disease resistance that have been elucidated in recent years and how these mechanisms could be used for the generation of improved plants resistant to CBB.     

Genetic mapping of resistance to bacterial blight disease in cassava (Manihot esculenta Crantz)

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease of cassava (Manihot esculenta Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F₁ cross (TMS 30572 × CM 2177–2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F₁ cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five Xam strains were used: CIO-84, CIO-1, CIO-136, CIO-295, and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9–20% of the phenotypic variance of the crop’s response to the five Xam strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.7–27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. Received: 20 September 1999 / Accepted: 30 December 1999     

导入双价基因的转基因杂交油菜亲本及其对菌核病抗性的研究

比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassica napus)的影响, 建立了油菜高效转化体系。按该体系, 将组成型表达几丁质酶和β-1, 3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系, 获得了转基因恢复系和保持系植株。对再生植株进行PCR和Southern Blot检测, 结果表明外源基因已整合到油菜基因组中。连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持, 证实外源基因已遗传到T3代。对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定, 结果表明, 转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高, 大田鉴定其发病率连续2年均比受体对照减少78%以上, 比抗病品种‘中油821’减少75%以上, 病情指数比受体对照和‘中油821’减少均达显著水平, 其抗病性能在后代稳定遗传, 获得了高抗菌核病的转基因育种材料。     

Relations Between Biomass Accumulation and Infection by Vesicular-Arbuscular Mycorrhizal Fungi in Cassava as Affected by Planting Date

Roots of 12, 3-month-old, field-propagated clones of cassava (Manihot esculenta Crantz) were more heavily mycorrhizal in the dry (Jan.–March and Oct.–Dec.) (39–83 %) than in the wet season (April–Sept.) (20–71%). Mean dry weights (biomass) of roots and shoots in the wet season (3.8–7.9 and 9.7–19.1 g/plant) were higher (P = 0.01) than in the dry season (2.1–5.9 and 5.4–12.8 g/plant, respectively). Clones with symptoms of the African cassava mosaic disease (ACMD) were less mycorrhizal, (20–69%) than mosaic symptom-free clones (51–83%). Higher colonization of roots of the clones by indigenous fungal symbionts and lower biomass accumulation in the dry season are attributed mainly to soil moisture and other effects, while reduced infections in cassava with ACMD symptoms may be due to ACMD-induced reductions in carbohydrate levels.     

The Effect of Temperature on Symptom Expression and Colonization in Resistant and Susceptible Carnation Cultivars Infected with Fusarium oxysporum f. sp. dianthi

Six commercial carnation cultivars were inoculated with Fusarium oxysporum f. sp. dianthi race 2, and grown under three different temperature regimes. Colonization by the pathogen and development of wilt symptoms were assessed at intervals up to 40 days. No symptoms and very little colonization were seen in any of the cultivars at 14–15°C. At a temperature of 22°C, the cultivars were clearly differentiated into three groups: those with resistance, partial resistance or susceptibility to the pathogen depending on the severity of symptoms and the extent of fungal colonization. Symptom severity was associated with the extent of colonization. This differentiation was not seen at 26°C, when all cultivars except the most resistant, cv.‘Carrier 929′, rapidly became diseased and died by 23 days after inoculation. ‘Carrier 929’ also showed some wilt symptoms at this temperature and was colonized throughout the height of the stem after 40 days. The pathogen caused disease at 26°C by a combination of vascular wilting and stem base and root rotting. Fungal colonization was assayed at 22°C by the dilution plate/homogenization method and by estimation of fungal chitin in a highly resistant (‘Carrier 929′) and in a highly susceptible (‘Red Baron’) cultivar. Both methods of assay gave similar results. In ‘Red Baron’, colonization increased slowly up to 20 days after inoculation then progressed rapidly, closely following the development of severe wilt symptoms. In ‘Carrier 929’, colonization remained very low. The low level of fungal biomass in ‘Carrier 929’ compared with ‘Red Baron’ indicated that the former cultivar showed true resistance as opposed to tolerance to the disease.     

Interaction of quantitative trait loci for resistance to common bacterial blight and pathogen isolates in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.

Common bacterial blight (CBB) is a major disease of common bean (Phaseolus vulgaris L.) worldwide. Genetic resistance is the most effective and environmentally safe approach for controlling CBB, and identification of resistance quantitative trait loci (QTL) can improve response to selection when breeding for CBB resistance. Interactions of CBB resistance QTL and pathogen isolates with different levels of aggressiveness were studied using an F _4:5 recombinant inbreed line (RIL) population, derived from a cross between the susceptible cultivar “Sanilac” and the resistant breeding line “OAC 09-3.” Disease phenotyping was performed under field and growth room conditions using multiple bacterial isolates with differential levels of aggressiveness. QTL analysis was performed with 237 molecular markers. The effect of pathogen isolate on the average phenotypic value in the RIL population and the interaction of RILs and the pathogen isolates were highly significant. Two QTL underlying CBB resistance were detected on Pv08 and Pv03. A major QTL (R ² _p between 15 and 56%) was identified in a 5-cM (380 kbp) interval in the distal end of the long arm of Pv08. This genomic region was significantly associated with multiple disease evaluation traits in field and growth room assays and against different isolates of the pathogen, which included the previously known CBB marker SU91. A new QTL on Pv03 (Xa3.3^SO), associated with the PvSNP85p745405 allele from the susceptible parent, Sanilac, appeared to be an isolate-specific QTL against the aggressive fuscans isolate ISO118. Interaction between the SU91 and Xa3.3^SO QTL resulted in a significant reduction in mean disease severity for almost all disease evaluation traits after plants were challenged with the isolate ISO118. The 7.92 and 7.79% diseased areas in RILs with both QTL, compared with 14.92 and 13.81% in RILs without either in test1 and in test2 quantified by image analysis, showed a 44 and 47% reduction of percent diseased areas, indicating that the two QTL interact to limit the expansion of CBB symptoms after infection by ISO118. The information obtained in this study indicates that while the broad-spectrum SU91 QTL is useful in breeding programs, isolate-specific QTL, such as Xa3.3^SO, will aid in breeding bean varieties with enhanced resistance against aggressive regional isolates.     

A blue dive: from ‘blue fingers’ to ‘blue silver’. A comparative overview of staining methods for in-gel proteomics

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of ‘blue silver’ colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and ‘blue silver’ CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the ‘blue silver’ method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented ‘blue silver’ can evidence the complexity of the sample.     

Effect of inoculation of symbiotic fungi on the growth and antioxidant enzymes’ activities in the presence of <Emphasis Type="Italic">Fusarium subglutinans</Emphasis> f. sp. <Emphasis Type="Italic">ananas</Emphasis> in pineapple plantlets

The inoculation with symbiotic fungi, Arbuscular mycorrhizal fungi (AMF) and/or Piriformospora indica on the growth, nutrient absorption, and induction of antioxidant enzyme activities in plantlets of pineapple ‘Imperial’ (fusariosis-resistant) and ‘Pérola’ (fusariosis-susceptible) in the presence of Fusarium subglutinans f. sp. ananas was investigated. The experiment was comprised by two cultivars, with or without fungal inoculation (Claroideoglomus etunicatum, Rhizophagus clarus, and P. indica, a mixture of all the fungi, and the control—absence of fungal inoculation), with or without applying Fusarium conidia, and with four replicates. In both cultivars, nutrient absorption was higher in the AMF plantlets compared to those inoculated with P. indica or the control ones, although it was more efficient in ‘Imperial’ than in ‘Pérola’. Inoculation with AMF and/or P. indica as well as the pathogen influenced differently the activities of superoxide dismutase, catalase, glutathione reductase, peroxidase, and polyphenol oxidase, in the shoots or roots of pineapple plantlets in both cultivars. Inoculated plantlets with mixture of all the fungi also exhibited a better growth and nutrient absorption, and generally, the ‘Imperial’ responded better than ‘Pérola’. In addition, these plantlets developed better than the control even in the presence of pathogen, indicating that inoculation with AMF and/or P. indica may contribute to the production of more resistant propagative material. Increased antioxidant enzyme activity is a potential strategy for managing this plant for explore biological control as an alternative to reduce environmental and health impacts by reducing the use of fungicides.     

Assessment of laboratory methods for evaluating cassava genotypes for resistance to root rot disease

Field evaluation of six cassava genotypes for resistance to root rot disease was compared with three rapid laboratory methods (whole root inoculation, root slice inoculation, and stem inoculation) for resistance screening. Both the field evaluation and the three laboratory methods separated the varieties into resistant and susceptible groups. Genotypes 30572 and 91/02324 were resistant while 92/0247, 92/0057 and TME-1 were susceptible. One genotype (30001) was not consistent in its reaction between field evaluation and laboratory assays. In the laboratory assays with three fungal pathogens, different pathogens varied in their levels of virulence on host genotypes. With the most virulent pathogen (Botryodiplodia theobromae), the majority of the genotypes reacted in the same way across trials with the root slice and whole root assays. Due to the good correlation between the whole root assay and the field results, we recommend this for the routine assessment of cassava resistance to root rot disease and for the analysis of virulence of pathogen isolates. However, because of the advantages in terms of economy of labour, space, time, quantity of root and inoculum required, the root slice assay could be used for the preliminary screening of large cassava accessions. The selected genotypes can then be further screened with the whole root inoculation method.     

Effect of Inoculum Density on Verticillium Wilt Incidence in Commercial Olive Orchards

Verticillium wilt, caused by Verticillium dahliae Kleb., is presently the most destructive disease of olive, particularly in Andalucía (southern Spain). ‘Picual’ and ‘Arbequina’ are the dominant cultivars being planted in Spain. Both cultivars are highly susceptible to the defoliating pathotype of V. dahliae when artificially inoculated by root‐dipping or stem injection. Conversely, ‘Arbequina’ is considered more resistant than ‘Picual’ based on field observations and farmer's experience. In this study, the differential reaction between of cultivars was confirmed by surveys of naturally infested orchards with different inoculum densities of the pathogen. The average percentage of affected olive trees of ‘Picual’ was 60.2%, while only 13.1% of trees of ‘Arbequina’ showed disease symptoms. Overall, the pathogen caused extensive wilting of branches and defoliation on the trees of ‘Picual’, whereas ‘Arbequina’‐infected trees showed chlorotic symptoms and slight defoliation. The relationship between inoculum density and disease incidence fit a logarithmic function for both cultivars. The percentage of affected trees of ‘Arbequina’ per year increased linearly (y = 0.3559x, R² = 0.5652, and P = 0.0195) with the inoculum density in the soil, whereas this relationship was not observed for the ‘Picual’. Planting density had no effect on disease incidence for any of the two cultivars.     

Biological control of the two-spotted spider mite Tetranychus urticae on strawberries by the predatory phytoseiid mite Typhlodromus pyri (Acari, Tetranychidae, Phytoseiidae)

The ‘Mikulov’ strain of the predatory mite Typhlodromus pyri Scheuten from south Moravian vineyards was released on cultivated strawberries infested with the two-spotted spider mite, Tetranychus urticae Koch. The strawberries were grown in field plantations and under glass. Typhlodromus pyri on vine shoots were successfully introduced into the field strawberry plantation but they produced no demonstrable control of the spider mites and they eventually declined in density with their prey. In contrast, T. pyri gave good control of spider mites in the glasshouse despite the occurrence of low humidity and water stress of the plants.     

Autophagy-related genes serve as heat shock protein 90 co-chaperones in disease resistance against cassava bacterial blight

Heat shock protein 90 (HSP90) is involved in plant growth and various stress responses via regulating protein homeostasis. Autophagy keeps cellular homeostasis by recycling the components of cellular cytoplasmic constituents. Although they have similar effects on cellular protein homeostasis, the direct association between HSP90 and autophagy signaling remains unclear in plants, especially in tropical crops. In this study, the correlation between HSP90 and autophagy signaling was systematically analyzed by protein–protein interaction in cassava, one of the most important economy fruit in tropic. In addition, their effects on plant disease response and underlying mechanisms in cassava were investigated by functional genomics and genetic phenotype assay. The potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex interacts with MeATGs and subsequently triggers autophagy signaling, conferring improved disease resistance to cassava bacterial blight (CBB). On the contrary, HSP90 inhibitor and autophagy inhibitor decreased disease resistance against CBB in cassava, and autophagy may be involved in the potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex-mediated multiple immune responses. This study highlights the precise modulation of autophagy signaling by potential MeHSP90.9-MeSGT1-MeRAR1 chaperone complex in autophagy-mediated disease resistance to CBB.     

亚洲柑橘木虱成虫和5龄若虫在感染黄龙病的柑橘上的取食行为及获菌效率比较

【目的】亚洲柑橘木虱Diaphorina citri是柑橘的毁灭性病害——黄龙病亚洲种‘Candidatus Liberibacter asiaticus’(‘C las’)的主要传播媒介。本研究的目的是明确木虱成虫和5龄若虫的取食行为、获菌效率是否有差异,以及寄主感染黄龙病是否对5龄若虫取食产生影响。【方法】利用直流型刺吸电位仪(DC-EPG Giga-4)记录柑橘木虱成虫和5龄若虫在携带黄龙病的酸橘Citrus reticulata cv.Sunki嫩梢上10 h的取食行为,用qPCR单头检测其获得黄龙病病原菌的效率,并比较5龄若虫在感病和健康植株嫩梢上的取食行为。【结果】柑橘木虱成虫与5龄若虫在感染黄龙病的酸橘上的取食行为有显著差异。5龄若虫比成虫更快地开始在韧皮部和木质部进行吸食,口针在韧皮部的总过程以及吸食时间显著长于成虫。此外,5龄若虫和成虫在EPG测定(同时饲菌)10 h后获菌率分别为37.5%和20.0%,若虫明显高于成虫。寄主植物感染黄龙病对5龄若虫的取食行为有一定的影响,表现在感病植株上的刺探次数、唾液分泌次数和韧皮部吸食次数都显著少于健康植株,而两者分泌唾液和韧皮部吸食时间没有显著差异。另外,在感病植株上首次韧皮部取食出现时间较在健康植株上要早。【结论】在同等时间下,柑橘木虱5龄若虫比成虫在感染黄龙病的酸橘上的取食能力更强、取食量更大、获菌率更高,原因可能是若虫需要更多的营养物质供其生长发育所致。寄主感染黄龙病对木虱5龄若虫的取食有利,使其更快地开始取食,而且更改取食位点次数变少,推测可能与黄龙病菌破坏了植物的防御有关。     

Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae

Xanthomonas phaseoli pv. manihotis (Xpm) and X. cassavae (Xc) are two bacterial pathogens attacking cassava. Cassava bacterial blight (CBB) is a systemic disease caused by Xpm, which might have dramatic effects on plant growth and crop production. Cassava bacterial necrosis is a nonvascular disease caused by Xc with foliar symptoms similar to CBB, but its impacts on the plant vigour and the crop are limited. In this review, we describe the epidemiology and ecology of the two pathogens, the impacts and management of the diseases, and the main research achievements for each pathosystem. Because Xc data are sparse, our main focus is on Xpm and CBB.