Current Distribution of Huanglongbing (citrus greening disease) in India as Diagnosed by Real‐Time PCR

The widespread occurrence of Huanglongbing (HLB) was recorded in sixteen citrus growing states of India using the real‐time quantitative PCR and the derived threshold cycle (Ct) value. All the commercially important citrus varieties of mandarin, sweet orange, lime and lemon, pummelo and Satkara were infected with ‘Candidatus Liberibacter asiaticus’, the bacterium associated with HLB. Ct values positive for HLB were found in all the states except Arunachal Pradesh. The primer–probe combination HLBas‐HLBr‐HLBp was found specific to Ca. L. asiaticus and do not exhibit any cross‐reactivity with other pathogenic residents of citrus.     

Surveillance of Microsporidia and Protozoan Pathogens in Pensacola Florida: A One‐year Study

Pathogens and the potential risk they present to public health in recreational waters are of continual public concern. The focus of this study was a year‐long sampling campaign to document the presence of Microsporidia and protozoan pathogens in the Bayou Texar waterway in Pensacola, Florida. We used biofilms as sentinel indicators for trapping pathogens in five different locations in Pensacola, Florida. Of the 34 biofilm samples, 16 were positive for pathogens. Of these samples, 13 were positive for Enchephalitozoon spp. (mostly E. cuniculi), 11 were positive for Enterocytozoon bieneusi, and two were positive for Cryptosporidium parvum. The data demonstrate that Microsporidia were easily recovered and primarily present in water during summer months.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

Symbiosis interruption in the olive fly: Effect of copper and propolis on Candidatus Erwinia dacicola

The relationship between Bactrocera oleae (Rossi 1790) and its endosymbiont Candidatus Erwinia dacicola is important to achieving effective control of the olive fly population in the field. This bacterium plays a crucial role in the life of B. oleae and is necessary for its fitness. Thus, in the absence of the endosymbiont, B. oleae wild populations in the field might decrease considerably. Copper is one of the most used antimicrobials for horticultural crops worldwide, and its efficacy against Ca. E. dacicola has been demonstrated in field trials. Propolis is another natural antimicrobial compound largely used for its activity in several fields. If propolis and copper prove to be efficient against wild populations of the endosymbiont B. oleae in the field, such a biological restraint might improve sustainable agriculture. We evaluated, under laboratory conditions, the effect of two different copper products (at two different concentrations, 5% and 20%) and propolis on the content of Ca. E. dacicola in the eggs and in the adult oesophageal bulbs of B. oleae. Bulbs were extracted twice, after 2 and 5 weeks of exposure. Real‐time PCR on the bulbs showed a reduction in Ca. E. dacicola content in flies treated with copper (at both 5% and 20%), and from the first to the second extraction, while flies treated with propolis showed an increment of the relative abundance of Ca. E. dacicola. Both copper products (5% and 20%) reduced the egg production after 2 and 5 weeks in comparison with the control and propolis treatments. Moreover, adult mortality was significantly higher with propolis compared with the other treatments. Thus, our results encourage further research in order to develop new tools for the control of the olive fly in the framework of an integrated pest management strategy.     

Development of a TaqMan Real‐Time Polymerase Chain Reaction Assay for Detection and Quantification of Fusarium oxysporum f. sp. lycopersici in Soil

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is an important disease of tomato. Pathogenicity and vegetative compatibility tests, although reliable, are laborious for the identification of FOL isolates and cannot efficiently quantify population densities of FOL in the soil. The objective of this study was to develop a rapid, sensitive and quantitative real‐time polymerase chain reaction (PCR) assay for detecting and quantifying FOL in soil. An inexpensive and relatively simple method for soil DNA extraction and purification was developed based on bead‐beating and a silica‐based DNA‐binding method. A TaqMan probe and PCR primers were designed using the DNA sequence of the species‐specific virulence gene SIX1, which is only present in isolates of FOL, not in isolates of other formae speciales or non‐pathogenic isolates of F. oxysporum. The real‐time PCR assay successfully amplified isolates of three races of FOL used in this study and quantified FOL DNA in soils, with a detection limit of 0.44 pg of genomic DNA of FOL in 20 μl of the real‐time PCR. A spiking test performed by adding different concentrations of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by the real‐time PCR assay and the concentration of conidia added. In addition, the real‐time PCR assay revealed a significant quadratic regression for a glasshouse experiment between disease severity and DNA concentration of FOL. The soil DNA extraction method and real‐time PCR assay developed in this study could be used to determine population densities of FOL in soil, develop threshold models to predict Fusarium wilt severity, identify high‐risk fields and measure the impact of cultural practices on FOL populations in soils.     

Strategies in herbivory by mammals revisited: The role of liver metabolism in a juniper specialist (Neotoma stephensi) and a generalist (Neotoma albigula)

Although herbivory is widespread among mammals, few species have adopted a strategy of dietary specialization. Feeding on a single plant species often exposes herbivores to high doses of plant secondary metabolites (PSMs), which may exceed the animal's detoxification capacities. Theory predicts that specialists will have unique detoxification mechanisms to process high levels of dietary toxins. To evaluate this hypothesis, we compared liver microsomal metabolism of a juniper specialist, Neotoma stephensi (diet >85% juniper), to a generalist, N. albigula (diet ≤30% juniper). Specifically, we quantified the concentration of a key detoxification enzyme, cytochrome P450 2B (CYP2B) in liver microsomes, and the metabolism of α‐pinene, the most abundant terpene in the juniper species consumed by the specialist woodrat. In both species, a 30% juniper diet increased the total CYP2B concentration (2–3×) in microsomes and microsomal α‐pinene metabolism rates (4‐fold). In N. stephensi, higher levels of dietary juniper (60% and 100%) further induced CYP2B and increased metabolism rates of α‐pinene. Although no species‐specific differences in metabolism rates were observed at 30% dietary juniper, total microsomal CYP2B concentration was 1.7× higher in N. stephensi than in N. albigula (p < .01), suggesting N. stephensi produces one or more variant of CYP2B that is less efficient at processing α‐pinene. In N. stephensi, the rates of α‐pinene metabolism increased with dietary juniper and were positively correlated with CYP2B concentration. The ability of N. stephensi to elevate CYP2B concentration and rate of α‐pinene metabolism with increasing levels of juniper in the diet may facilitate juniper specialization in this species.