Effect of <Emphasis Type="Italic">Metarhizium guizhouense</Emphasis> infection on mating competition and mate choice of <Emphasis Type="Italic">Bactrocera latifrons</Emphasis> (Diptera: Tephritidae)

Metarhizium guizhouense PSUM02 treated males of Bactrocera latifrons were investigated for the mating competition among males and mating choice by female flies to develop an auto-dissemination for the control of B. latifrons. In the present study, on day 1–4 of experiment, M. guizhouense–treated male flies were equally competitive with the normal male flies as we did not observe any differences in mating by treated and normal male flies of B. latifrons. Further, mating competitiveness were found low in treated adult male B. latifrons than normal male B. latifrons from 5th days of treatment until death. Kaplan-Meier survival analysis of treated male flies gave average survival times (AST) of 4.3?±?0.1 days, while the healthy female and male flies in the same cage showed AST of 9.3?±?0.3 and 8.3?±?0.4 days, respectively. The AST of untreated flies in control experiment ranged from 14.2–14.5 days. In mating preference experiment, M. guizhouense–treated male flies were chosen by virgin female than gravid female flies for mating. The treated male flies caused mortality in both virgin and gravid female flies in the same cage with AST of 4.4?±?0.1, 5.6?±?0.1 and 7.4?±?0.2 days, respectively, while untreated flies showed AST ranged from 13.9–14.3 days in control. The treated male flies could transmit the fungus infection to both untreated female and male flies as well as in virgin and gravid female flies by mating and contact. Our experiments showed the potentiality of M. guizhouense PSUM02 in management of B. latifrons by auto-dissemination with treated male flies, which transmit the fungus to a healthy population to reduce insect pest infestations.     

Feeding potential of <Emphasis Type="Italic">Cryptolaemus montrouzieri</Emphasis> against the mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis>

Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is an exotic species native to the USA, damaging cotton and other plant families. The feeding potential of different development stages of Cryptolaemus montrouzieri Mulsant, a biological control agent against mealybugs, was investigated on different development stages of P. solenopsis. Fourth instar grubs and adults of C. montrouzieri were the most voracious feeders on different instars of mealybug. The number of 1^st instar nymphs of mealybug consumed by 1^st, 2^nd, 3^rd and 4^th instar larvae and adult beetles of C. montrouzieri was 15.56, 41.01, 125.38, 162.69 and 1613.81, respectively. The respective numbers of 2^nd and 3^rd instar nymphs of mealybug consumed were 11.15 and 1.80, 26.35 and 6.36, 73.66 and 13.32, 76.04 and 21.16, 787.95 and 114.66. The corresponding figures for adult female mealybugs were 0.94, 3.23, 8.47, 12.71 and 73.40, respectively. The results indicate that C. montrouzieri has the potential to be exploited as a biocontrol agent in North India; inoculative releases of 4^th instar larvae and adults may provide instant control of P. solenopsis. Field experiments should be conducted to determine the efficiency of the ladybird on this mealybug.     

Advanced lentil lines screened for resistance to <Emphasis Type="BoldItalic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="BoldItalic">lentis</Emphasis> under greenhouse and field conditions

Fusarium oxysporum f. sp. lentis is the most important pathogen of lentil plants, and most areas under lentil cultivation are reported to have a fusarium wilt disease background. The plants are infected in the seedling stage and later stages of their development. Fusarium wilt disease, which has appeared at high incidence rates during recent years, has caused sharp drops in the yield, especially in Moghan, in the northwest of Iran. Forty-five isolates of the pathogen were collected from different regions of the country with two isolates from ICARDA in the summer of 2008 and identified using Nelson’s key. The pathogenicity of the collected isolates was studied on a sensitive line (ILL 4605) under greenhouse conditions and significant differences in pathogenicity were found among them. The most pathogenic isolates from three provinces, East Azerbaijan (EA 30), Ardebil (Ar 3) and Khorasan (Kh 45), were selected and used in screening of 55 developed lines under greenhouse and field conditions. In the greenhouse, test plants were inoculated by immersing root tips in spore suspension and sowing seeds in pre-infested pot soil. Field tests were carried out in a naturally highly infested farm. At all stages, the plant response to the disease was based on the percentage of dead plants. Cluster analyses of the greenhouse and field data led to the selection of three lines (81S15, FLIP2007-42 L and FLIP2009-18 L) that were resistant under greenhouse and field conditions.     

Three genes of miraculin-like proteins from Nicotiana benthamiana with dissimilar putative structures show highly similar patterns of induction following bacterial and fungal infections

Expression of three Nicotiana benthamiana miraculin-like protein genes, NbMLP1, NbMLP2 and NbMLP3, showed almost identical responses to wounding, an incompatible interaction with Pseudomonas syringae pv. tabaci and compatible interactions with P. syringae pv. tabaci, Colletotrichum destructivum or Colletotrichum orbiculare. However, only NbMLP1 expression responded to exogenous methyl jasmonate or ethylene. None exhibited expression in healthy leaves and stems, and all showed highest expression in seeds, except for NbMLP1, which had highest expression in roots. NbMLP1, NbMLP2 and NbMLP3 were in different subfamilies of miraculin-like protein sequences of N. benthamiana and Nicotiana tabacum. Subfamilies correlated well with predicted features of the reactive-site loop potentially affecting the bond that could react with serine proteinases. Despite considerable predicted structural diversity that might affect biological activity, the apparently coordinated expression of these genes to pathogen attack may reflect the need to produce diverse proteinase inhibitors to act against a potentially broad range of secreted microbial proteinases during basal resistance to pathogens.     

Isotope-edited nuclear magnetic resonance: novel methodologies for investigating metabolism

While the use of NMR and stable isotopes in metabolism studies is hardly new, it is only recently that isotope-edited NMR spectroscopy has been applied in kinetic studies of glyphosate metabolism of soil microbes. NMR can detect multiple species simultaneously and non-destructively, yielding valuable information on structural identification of metabolites. T riple R esonance I sotope ED ited spectroscopy (TRIED), [²H]NMR, and [²H–¹³C] INEPT (I nsensitive N ucleus E nhancement through P olarization T ransfer) are three isotope-edited techniques which have been used in combination to examine the microbial degradation of glyphosate (N-phosphonomethylglycine). Using ¹³C- and ¹⁵N-labeled glyphosate, TRIED can detect multiple metabolites in crude matrices at submicrogram levels, an improvement over earlier techniques where milligrams were needed. It can detect 500 nanograms of ¹³C–¹⁵N-labeled compound in a crude sample (1 : 1400 mass ratio), only a few hours work being required. [²H]NMR and [²H–¹³C]INEPT were also used as complementary techniques to further examine metabolites whose ¹³C–¹⁵N bond has been cleaved. The three-isotope-edited methods produced results consistent with both radioactivity and HPLC analyses. Accordingly, we are able to detect minute levels of metabolites in the presence of complex mixtures, minimizing the costs and time of sample purification. ©1999 Society of Chemical Industry     

Seasonal changes in germination response of buried seeds of Orobanche crenata Forsk.

Orobanche crenata seeds, collected in Syria, Egypt and Spain, were buried in the field in Syria (all three seed lots) and Spain (only Spanish seeds) and at regular intervals exhumed and tested for germination, to investigate whether the seeds exhibit an annual dormancy/non-dor- mancy cycle. When exposed directly to the synthetic germination stimulant GR24 for 7 days at 20°C, seeds only germinated in autumn after the first rains and to a limited extent in winter. When the seeds were conditioned for 11 days at 20°C prior to exposure to GR24, germination occurred during summer and autumn, but seeds were dormant in winter and early spring. The observed seasonal pattern in germinability, in relation to rainfall and soil temperature, was largely consistent with the results of an in vitro experiment by Van Hezewijk et al. (1993), investigating the effect of conditioning temperature and conditioning period on germination capacity and the development of secondary dormancy. Moisture and temperature can therefore be considered the major factors regulating induction and alleviation of dormancy in buried O. crenata seeds. There were no basic differences in response owing to site of collection of O. crenata seeds, nor to the location where they were buried. Variations saisonnières des exigences de germination de graines enfouies d'Orobanche crenata Forsk. Des graines d'Orobanche crenata récoltées en Syrie, en Égypte et en Espagne ont été enfouies au champ en Syrie (les 3 lots) et en Espagne (seules les graines d'Espagne) puis ont été exhumées a intervalles régulier pour que leur aptitude à la germination soil évaluée. Le but était de déterminer si les graines possédaient un cycle annuel dormance/non dormance. Quand elles étaient directement exposées au stimulant de germination synthétique GR24 pendant 7 jours à 20°C, les graines ne germaient qu'à l'automne après les premières pluies et peu en hiver. Quand les graines restaient pendant 11 jours à 20°C avant leur exposition au GR24, la germination seproduisait en été et à l'automne mais les graines restaient dormantes en hiver et au début du prin-temps. Les variations saisonnières d'aptitude à la germination, liées aux précipitations et à la temperature du sol, étaient en accord avec les résultats d'une expérience in vitro de Van Hezewijk et al. (1993) concernant l'effet de la température et de la durée pendant laquelle elle est appliquée, sur l'aptitude à la germination et le développement de la dormance secondaire. L'humidité du sol et sa température peuvent ainsi être considérées comme les principaux facteurs qui induisent et lèvent la dormance de graines de O. crenata enfouies. On n'observait pas de différences importantes dues au lieu de récolte ou à l'endroit oü elles étaient enfouies. Jahreszeitliche Änderungen der Keimung von vergrabenen Samen von Orobanche crenata Forsk. Proben von in Syrien, Ägypten und Spanien gesammelten Orobanche-crenata-Samen wurden in Syrien und Proben nur spanischer Herkunft in Spanien im Freiland im Boden ausgelegt und in regelmäßigen Zeitabständen ausgegraben und auf ihre Keimfähigkeit getestet, um zu untersuchen, ob die Samen einen jährlichen Dormanz-Zyklus haben. Beim direktem Auslegen in dem synthetischen Keimungsmittel GR24 öber 7 d bei 20°C keimten die Samen nur im Herbst nach den ersten Regenfällen und in beschränktem Umfang im Winter. Wenn die Samen för 11 d bei 20°C vor dem Auslegen in GR24 vorbehandelt worden waren, keimten sie im Sommer und Herbst, aber im Winter und fröhen Fröhjahr waren sie dormant. Das jahreszeitliche Verhalten der Keimfähigkeit in Abhängigkeit von Niederschlag und Bodentemperatur stimmte weitgehend mit den Ergebnissen eines In-vitro-Versuches von Van Hezewijk et al. (1993) öber die Wirkung einer Wärmevorbehandlung und Vorbehandlungszeit auf die Keimfähigkeit und die Ausprägung sekundärer Dormanz öberein. Bodenfeuchte und -temperatur können deshalb als die wichtigsten Faktoren för die Induktion und Aufhebung der Dormanz von Orobanche-crenata-Samen im Boden angesehen werden. Herkunft und Versuchsort hatten keinen erheblichen Einfluß auf die Ergebnisse.     

Outbreak of MAV-type barley yellow dwarf virus on wheat in the Garhwal Hills in India

A serious outbreak of barley yellow dwarf luteovirus (BYDV, MAV-type) on wheat in the Garhwal Hills, Central Himalayas, India is reported. This is the first conclusive evidence based on serology for the presence of MAV-BYDV in India.     

Pathogenicity and mycotoxin production by <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> isolated from onion and garlic in Serbia

Fusarium proliferatum can occur on a wide range of economically important vegetable plants but its role in disease is not always well established. In 2000 and 2001, from forty-one field samples of wilting onion and garlic plants in Serbia, F. proliferatum as the predominant fungal species was isolated from root and bulbs. Seventy isolates were firstly characterized for their sexual fertility and were shown to be mostly members of Gibberella intermedia (sixty-seven of seventy isolates, the remaining three isolates were unfertile), the sexual stage of F. proliferatum (syn. mating population D of G. fujikuroi complex). A selected set of eleven F. proliferatum isolates from both hosts were also tested for their pathogenicity and toxigenicity. Although onion and garlic plants were susceptible to all isolates, onion plants showed a significantly higher disease severity index. Six of the eleven isolates of F. proliferatum produced fumonisin B₁ from 25 to 3000 μg g⁻¹, and beauvericin from 400 to 550 μg g⁻¹; ten isolates produced fusaric acid from 80 to 950 μg g⁻¹ and moniliformin from 50 to 520 μg g⁻¹. Finally, all isolates produced fusaproliferin up to 400 μg g⁻¹. These results confirm F. proliferatum as an important pathogen of garlic and onion in Europe and that there is a potential mycotoxin accumulation risk in contaminated plants of both garlic and onion.     

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

An enrichment microsphere immunoassay for the detection of <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> in potato tuber extracts

An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100–1000 cfu ml^?1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 10⁶ and 10⁷ cells ml^?1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.