Transmission of a sugarcane yellow leaf phytoplasma by the delphacid planthopper Saccharosydne saccharivora, a new vector of sugarcane yellow leaf syndrome

During surveys of sugarcane fields in western and central Cuba from December 2001 to March 2003, the delphacid planthopper Saccharosydne saccharivora was the most prevalent of the Auchenorrhyncha fauna surveyed. Individuals of S. saccharivora collected tested positive for the sugarcane yellow leaf phytoplasma (SCYLP). Saccharosydne saccharivora were reared in cages and used for experimental transmission studies of SCYLP. The S. saccharivora were given acquisition-access feeds of 72 h on SCYLP-infected canes collected from the field followed by an inoculation-access period of 15 days on healthy sugarcane seedlings. Symptoms of yellow leaf syndrome developed on 24 out of 36 plants, 7–12 months postinoculation. None of the 36 healthy seedlings that were inoculated with S. saccharivora fed on phytoplasma-free sugarcane developed symptoms. All phytoplasma-positive sugarcane and S. saccharivora samples showed identical RFLP patterns and had 99·89% similarity in their 16S/23S spacer-region sequences, but only 92·6–93·6% similarity with other phytoplasmas. Sequences were deposited with GenBank [accession numbers: AY725237 ( S. saccharivora ) and AY257548 (sugarcane)]. Phylogenetic analysis suggested that the phytoplasmas from sugarcane and S. saccharivora are putative members of a new 16Sr phytoplasma group. This is the first report of vector transmission of a phytoplasma associated with sugarcane yellow leaf syndrome and the first time that S. saccharivora has been shown to vector a phytoplasma.     

Systemic infection with potato virus YNTNalters the structure and activity of the shoot apical meristem in a susceptible potato cultivar

Stem nodes of the potato virus Y^NTN(PVY^NTN) susceptible potato cultivar (Solanum tuberosum cv. “Igor”) were studied to investigate whether alterations in shoot morphology caused by infection with PVY^NTNare related to changes in structure and activity of the shoot apical meristem. The PVY^NTNcauses severe disease symptoms known as the potato tuber necrotic ringspot disease (PTNRD) in susceptible potato plants grown in the field. Although in stem node culture the symptoms are mildly expressed, in systemically PVY^NTN-infected plants the shoot height stem diameter, number of leaves and the total leaf area were lower than in the healthy control plants at day 18 and 35 of culture. Histological analysis of the longitudinal sections of the shoot tip revealed that the infected plants had smaller shoot apical meristems due primarily to a lower cell number in different meristem zones. One of the most affected zones of the apical shoot meristem was the peripheral zone, the region in which leaf primordia are induced. The data is consistent in showing that the infection of potato plants with PVY^NTNleads to alterations in the shoot apical meristem structure and mitotic activity. These changes are, in turn, reflected in altered overall morphology of the infected plant.     

Double-edged effects of the cryogenic technique for virus eradication and preservation in shallot shoot tips

Plant virus eradication is a prerequisite for the use of virus-free propagules for sustainable crop production. In contrast, virus preservation is required for all types of applied and basic research of viruses. Shoot tip cryopreservation can act as a double-edged strategy, facilitating either virus eradication or virus preservation in cryoderived plants. Here, we tested the efficacies of shoot tip cryopreservation for virus eradication and preservation in shallot (Allium cepa var. aggregatum). In vitro stock shallot shoots infected with onion yellow dwarf virus (OYDV) and shallot latent virus were thermotreated for 0, 2, and 4 weeks at a constant temperature of 36℃ before shoot tip cryopreservation. Results showed that viruses were preserved in recovered shoots when thermotherapy was not applied. Although thermotherapy lowered the regrowth levels of cryotreated shoot tips, the efficiency of virus eradication increased from 5% to 54%. Immunolocalization of OYDV and histological observation of cryotreated shoot tips showed the high frequency of virus preservation was due to the viral invasion of cells close to the apical meristem and the high proportion of cells surviving. Four weeks of thermotherapy drastically decreased the distribution of OYDV, as well as the percentage of surviving cells within the shoot tips, thereby promoting virus eradication. Virus-free plants obtained from combining thermotherapy with cryotherapy showed significantly improved vegetative growth and bulb production. The present study reports how thermotherapy can act as a trigger to facilitate either the safe preservation of Allium viruses or the production of virus-free shallot plants.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants grown in the field

Interaction between phytoplasma and grapevine at the physiological level is still poorly understood, as are plant defence mechanisms against the pathogen. This study investigates the level of gene expression of three selected genes in a large number of grapevine plants belonging to six disease/cultivar groups (healthy Chardonnay, Bois noir-infected Chardonnay, Flavescence dorée-infected Barbera and Prosecco, and recovered Barbera and Prosecco). All plants were grown in vineyards in uncontrolled conditions in order to represent the physiology of disease as accurately as possible. Sucrose synthase was significantly upregulated in infected plants of all cultivars with the lowest P -values in cvs Chardonnay and Prosecco ( P  < 0·001) and median fold-change around 2. This clearly indicates that carbohydrate metabolism changed in infected compared to healthy or recovered plants. Alcohol dehydrogenase I was significantly upregulated in infected relative to healthy Chardonnay plants ( P  < 0·05) indicating that alcoholic fermentation, a sign of hypoxic conditions, was induced in infected plants. Heat shock protein 70 was upregulated in infected compared to recovered plants only in cv. Prosecco. Linear discriminant analysis showed that classification of samples into disease status groups based on gene expression was highly accurate (82%), indicating that the response of field-grown plants to phytoplasma infection at the level of expression of selected genes was so intensive and uniform that it was possible to detect it in grapevine plants regardless of natural variables.     

Seed transmission of maize downy mildew (Peronosclerospora sorghi) in Nigeria

In an area of Nigeria where downy mildew of maize is present, histological assessment of maize seed revealed the presence of mycelium and oospores of Peronosclerospora sorghi in the kernels. Seed transmission of downy mildew of maize was demonstrated when grain purchased at local markets gave mean seedling infection rates of 12·3% (untreated seeds) and 10·0% (in metalaxyl-treated seeds) within 7 days of emergence, after storage in a desiccator for 30 days. When untreated seeds taken from nubbin ears of systemically infected plants from four states in southern Nigeria were planted at 9 days (17–22% moisture content) and 27 days (9–22% moisture content) after harvest, 20·0% infected seedlings resulted in both trials. Seeds from Borno state in northern Nigeria had 26·6% systemic seedling infection after 9 months of storage at 11% moisture content. When seeds harvested from maize plants inoculated with P. sorghi through silks were examined histologically, hyphae of P. sorghi were observed mostly in the scutellum of the embryo. Transmission of disease to seedlings was observed when the silk-inoculated seeds (9% moisture content) were planted in pots in a greenhouse; however, no disease transmission was observed when such seeds were planted in the field. The epidemiological significance of seed transmission is discussed with particular reference to survival of inoculum and development of epidemics. Also noteworthy is the overall significance of seed transmission in Nigeria, where the major source of seed is that saved by farmers from their grain crop, occasionally supplemented by seed bought from the local market.     

Translocation of 14C-labelled photoassimilates to roots in barley: effects of mildew on partitioning in roots and the mitotic index

First leaves of barley seedlings grown in solution culture were inoculated with powdery mildew when the second leaf was fully emerged. ¹⁴C-labelled carbon dioxide was fed to either first or second leaves of infected and non-infected plants. Translocation of labelled photoassimilates into roots, and partitioning into soluble, storage and structural fractions were studied in tip, mid and basal regions of primary roots 24 h after feeding. Mildew reduced the total activity in the plant, but had little effect on assimilate distribution since the percentage activity translocated to roots was only transiently reduced, approximately 7 days after infection. Reduced import led to a reduction in the specific activity of different fractions within roots, reductions being greatest in root tips. These changes were similar whichever leaf was fed. Changes in specific activity became progressively more pronounced in the 10 days following infection, and were paralleled by a reduction in the mitotic index of root tips. It is concluded that meristematic activity, and thus the growth, of primary roots of barley is particularly sensitive to reductions in photoassimilation caused by powdery mildew infection.     

Effects of bean rust (Uromyces appendiculatus) epidemics on host dynamics of common bean (Phaseolus vulgaris)

Epidemics of bean rust ( Uromyces appendiculatus ) and their effects on host dynamics of common bean ( Phaseolus vulgaris ) were studied in three controlled greenhouse experiments, with and without fungicide sprays, on two susceptible bean cultivars, Dufrix and Duplika. Bean plants were artificially inoculated with a suspension of 10⁵ U. appendiculatus urediniospores mL⁻¹ water and temporal disease progress, as well as host growth dynamics (leaf size and defoliation), were monitored on a leaflet basis in comparison with non-inoculated plants, which were sprayed with deionized water. Progress curves of bean rust, expressed as the proportion of leaf area occupied by pustules (uredinia), or as the proportion occupied by total lesion area (= halo areas + pustule area), were well described by logistic functions with maximum disease levels clearly lower than 1. Bean rust epidemics substantially affected host growth by reducing the total leaf area formed by 17·4–35·6% and 35·3–46·2% compared with healthy plants for cvs Dufrix and Duplika, respectively. Fungicide sprays mitigated the negative effect of bean rust, leading to a gain in leaf area of 17–21% compared with unsprayed plants in both cultivars in two experiments, while in another experiment, disease control had no effect in Dufrix, but a clear effect in Duplika. In addition to the growth depression, bean rust also led to pronounced losses of leaf area as a result of reduced leaf size (leaf shrivelling) and accelerated defoliation.     

Biology of a new virus isolated from Lupinus nootkatensis plants in Alaska

A new virus named Nootka lupine vein-clearing virus (NLVCV) was isolated from Lupinus nootkatensis plants that were confined to a relatively small area in the Talkeetna mountains of south-central Alaska. Annual surveys (2000–03) consistently found leaf symptoms of pronounced vein clearing and mosaic on 3- to 4-week-old plants in late June. Spherical particles ≈30 nm in diameter were isolated from these leaves. Virions contained a single-stranded RNA of ≈4·0–4·2 kb and one species of capsid protein estimated to be ≈40 kDa. The double-stranded RNA profile from naturally infected leaves consisted of three major bands ≈4·2, 1·9 and 1·5 kbp. Protein extractions from either sap or virions of diseased plants reacted to polyclonal antiserum made against the virions in Western blot assays. A predicted PCR product ≈500 bp was synthesized from virion RNA using primers specific to the carmovirus RNA-dependent RNA polymerase (RDRP) gene. The nucleotide sequence of the amplified DNA did not match any known virus, but contained short regions of identity to several carmoviruses. Only species belonging to the Fabaceae were susceptible to NLVCV by mechanical inoculation. Based on dsRNA profile, size of virion RNA genome and capsid protein, and similarity of the RDRP gene to that of other carmoviruses, it is suggested that NLVCV is a member of the family Tombusviridae , and tentatively of the genus Carmovirus . As the host range, RDRP gene and dsRNA profile of NLVCV are different from those of known viruses, this is a newly described plant virus.     

Attempts to eliminate <Emphasis Type="Italic">Candidatus</Emphasis> phytoplasma phoenicium from infected Lebanese almond varieties by tissue culture techniques combined or not with thermotherapy

Elimination of Candidatus phytoplasma phoenicium from two infected Lebanese varieties of almond by using different tissue culture techniques is reported. Except for the oxytetracycline therapy which totally inhibited the development of explants, stem cutting cultures associated with thermotherapy, shoot tip cultures associated or not with thermotherapy, and shoot tip micrografting were all suitable, either for shoot regeneration or for elimination of phytoplasma from the two varieties. However, stem cutting culture coupled with thermotherapy seemed to be the most effective for regeneration of phytoplasma-free plantlets.     

Infection and colonization of melon roots by Monosporascus cannonballus in two cropping seasons in Arizona and California

Although canopy collapse of melons (one of the above-ground symptoms of vine decline caused by Monosporascus cannonballus ) occurred late in the growing season, the onset of root infection occurred much earlier. In three early winter-spring and two late winter-spring crops, the onset of root infection occurred 47–65 and 35–36 days after planting, respectively. In contrast, in four summer-autumn crops, the onset of root infection occurred within 9–17 days after planting. Vine decline occurred commonly in winter-spring crops, but did not occur in any of the summer-autumn crops. Following the onset of root infection, the percentage of plants infected increased at rates of 0·031–0·036 and 0·038–0·070 per unit per day for winter-spring and summer-autumn crops, respectively, based on the monomolecular disease progress model. Root lesions were first observed 14–42 days after the onset of infection in winter-spring crops, and 14–28 days after the onset of infection in summer-autumn crops. Pathogen reproduction occurred primarily at the end of each growing season.     

水稻橙叶病分子检测及其在华南地区的发生与分布研究

对华南地区水稻橙叶病(rice orange leaf disease)进行病原菌分子检测和病情发生与分布调查。结果表明,该病是一种由电光叶蝉(Inazuma dorsalis)和黑尾叶蝉(Nephotettix cinticeps)传播的植原体(Phytoplasma)引起的病害,早期感病植株在分蘖期全株叶片表现为橙黄色,不久干枯死亡,对水稻生产造成严重为害。电镜下观察,病株幼叶叶脉筛管细胞中存在大量植原体。巢式PCR可从来源于水稻病株及介体电光叶蝉和黑尾叶蝉DNA抽提物中获得单一扩增产物,PCR产物核苷酸序列与文献报道的水稻橙叶植原体16S r DNA序列(Gen Bank登录号:KR061356)同一率均为99.8%以上。2015年广东各主要稻区及海南中部和广西东南部均有该病发生与分布。     

A phytoplasma representative of a new subgroup, 16SrXIII-E, associated with Papaya apical curl necrosis

Papaya apical curl necrosis (PACN) has frequently been observed in several Brazilian states. Affected plants exhibit foliar chlorosis, curvature of the apex, shortening of the internodes leading to bunching of the crown leaves, necrosis of the young apical parts, leaf drop, and dieback. Naturally infected plants were sampled and subjected to PCR assays, which confirmed that a phytoplasma was associated with the disease. Sequencing of the 16S rRNA gene, conventional and computer-simulated RFLP analyses, and phylogenetic analysis allowed the determination of the PACN phytoplasma as a representative of a new subgroup, designated 16SrXIII-E. The phytoplasmas of various 16Sr groups, including 16SrI, 16SrII, 16SrX, 16SrXII, and 16SrXVII, are known to be involved in anomalies in papaya plants in several countries. However, the present study reports, for the first time, the occurrence of a 16SrXIII phytoplasma in association with a papaya disease.     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Quantitative relationships between inoculum of Melampsora larici-epitea and corresponding disease on Salix

Six Salix clones were inoculated with urediniospores of four isolates of Melampsora larici-epitea at five inoculum levels using a leaf-disc method. Disease reactions were recorded using a digital camera; the number and size of uredinia were examined using image analysis software; and spore yield per leaf disc was measured. In three Salix / Melampsora combinations, S.  ×  mollissima 'Q83'/Q1 (LET4); S. viminalis '78183'/V1 (LET1); and S.  ×  stipularis /V1, pustule numbers increased as inoculum density became higher. In the remainder, S. viminalis 'Mullatin'/V1; S.  ×  calodendron /DB (LET3); and S. burjatica 'Korso'/K (LR1), pustule numbers initially increased, then decreased as inoculum densities exceeded 140–360 spores per disc. Calculated infection efficiency ranged from 0·11 to 0·20 on the three willows inoculated with V1: 0·16–0·68 for S.  ×  calodendron /DB; 0·20–0·55 for 'Q83'/Q1; and 0·07–0·48 for Korso/K. In single-spore inoculations, up to 10% of spores produced single uredinia. Infection efficiency increased sharply between inoculum densities of 1–40 spores per leaf disc. Spore yield was more closely correlated to pustule area (accounting for 61·2% variance for the combined data) than to the number of pustules (42·7% variance). For spore yields in relation to pustule numbers, clone-specific individual lines having different intercepts and slopes fitted significantly better than either a single line for all the tested willows, or parallel lines fitted to each clone ( P  < 0·001). For spore yields in relation to pustule area, clone-specific individual parallel lines were significantly better than a single line ( P  < 0·001).     

Sensitive Detection of a Phytoplasma Associated with Little Leaf Symptoms in Withania somnifera

Withania somnifera is an important medicinal plant native to the Indian-sub continent. Owing to the presence of a number of precious alkaloids, flavonoids and withanolides, it is widely used in the Indian and African systems of medicines. It is severely affected by phytoplasma present in the sieve tubes of phloem. With a view to micropropagate phytoplasma-free W. somnifera plants, an efficient and effective nested PCR-based system was developed for detection of associated phytoplasmas. Universal primers, designed from the 16S rDNA sequences of phytoplasmas, were applied in direct/nested-PCR. Total DNA extracts from leaf tissues of 33 suspected symptomatic and 11 non-symptomatic plants were subjected to direct PCR. The direct PCR products were subsequently employed as templates in nested PCR. The nested PCR could reamplify direct PCR products yielding a DNA fragment of 1.4 kb. A phytoplasma was detected in all the diseased plants and not from the healthy looking plants. Further, it was sensitive enough to amplify phytoplasma DNA obtained from crude DNA diluted up to 2500 times from naturally infected plants and also from various stages of in vitro-propagated diseased plants. Identical restriction fragment polymorphism enzyme profiles were obtained following restriction enzyme digestion of nested PCR products, obtained from five different plants, by EcoRI, AluI and RsaI restriction endonucleases. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro-generated plants and probably identification of, as yet unknown, hosts and vectors of phytoplasma associated with phytoplasma disease of W. somnifera.     

Shoot tip cryotherapy for plant pathogen eradication

Diseases caused by plant pathogens such as viruses, viroids and phytoplasmas cause huge economic losses of agricultural production and limit the safe movement of plant materials across borders. The use of pathogen-free planting materials provides a strategy for efficient management of these diseases and facilitates the global exchange of genetic resources. Shoot tip cryotherapy is a novel biotechnology method that uses cryogenic procedures to eradicate plant pathogens from the diseased plants. Combining thermotherapy or chemotherapy with shoot tip cryotherapy has further enhanced pathogen eradication efficiency. This review provides updated and comprehensive information on shoot tip cryotherapy and the combination of thermotherapy or chemotherapy with shoot tip cryotherapy for pathogen eradication. Prospects are proposed for future studies.     

A new root-knot nematode, Meloidogyne silvestris n. sp. (Nematoda: Meloidogynidae), parasitizing European holly in northern Spain

High infection rates of European holly ( Ilex aquifolium ) feeder roots by an unknown root-knot nematode were found in a holly forest at Arévalo de la Sierra (Soria province) in northern Spain. Holly trees infected by the root-knot nematode showed some decline and low growth. Infected feeder roots were distorted and showed numerous root galls of large (8–10 mm) to moderate (2–3 mm) size. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees of sequences within the ribosomal DNA (rDNA) demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European holly plants, but did not reproduce on a number of cultivated plants, including tomato, grapevine, princess-tree and olive. The species is described here, illustrated and named as Meloidogyne silvestris n. sp. The new root-knot nematode can be morphologically distinguished from other Meloidogyne spp. by: (i) roundish perineal pattern, dorsal arch low, with fine, sinuous cuticle striae, lateral fields faintly visible; (ii) female excretory pore level with stylet knobs, or just anterior to them, EP/ST ratio about 0·8; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anterior to excretory pore and short, sub-digitate tail; and (iv) males with lateral fields composed of four incisures, with areolated outer bands. Phylogenetic trees derived from maximum parsimony analysis based on 18S, ITS1-5·8S-ITS2 and D2–D3 of 28S rDNA showed that M. silvestris n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. ardenensis , M. dunensis and M. lusitanica .     

Size, shape and intensity of aggregation of take-all disease during natural epidemics in second wheat crops

Point pattern analysis (fitting of the beta-binomial distribution and binary form of power law) was used to describe the spatial pattern of natural take-all epidemics (caused by Gaeumannomyces graminis var. tritici ) on a second consecutive crop of winter wheat in plots under different cropping practices that could have an impact on the quantity and spatial distribution of primary inoculum, and on the spread of the disease. The spatial pattern of take-all was aggregated in 48% of the datasets when disease incidence was assessed at the plant level and in 83% when it was assessed at the root level. Clusters of diseased roots were in general less than 1 m in diameter for crown roots and 1–1·5 m for seminal roots; when present, clusters of diseased plants were 2–2·5 m in diameter. Anisotropy of the spatial pattern was detected and could be linked to soil cultivation. Clusters did not increase in size over the cropping season, but increased spatial heterogeneity of the disease level was observed, corresponding to local disease amplification within clusters. The relative influences of autonomous spread and inoculum dispersal on the size and shape of clusters are discussed.     

Viruses of Colocasia esculenta and Xanthosoma saggitifolium

Abstract

Bacterial blight of cassava is a serious problem in Central and South America and has been observed in parts of Africa. Symptoms include leaf spotting, wilting, die-back, gum exudation on young shoots, and vascular discoloration in mature stems and roots of susceptible cultivars. Dispersal by rain splashing is the most important means of dissemination within localized areas. Dissemination from one area to another occurs through infected planting material or by the use of contaminated tools. Delay in spread of the disease has been obtained by pruning infected plants. The use of resistant varieties and the production of certified bacteria-free planting material, obtained from plants propagated from shoot tip cuttings, has given satisfactory control.