The Red Queen lives: Epistasis between linked resistance loci

A popular theory explaining the maintenance of genetic recombination (sex) is the Red Queen Theory. This theory revolves around the idea that time‐lagged negative frequency‐dependent selection by parasites favors rare host genotypes generated through recombination. Although the Red Queen has been studied for decades, one of its key assumptions has remained unsupported. The signature host‐parasite specificity underlying the Red Queen, where infection depends on a match between host and parasite genotypes, relies on epistasis between linked resistance loci for which no empirical evidence exists. We performed 13 genetic crosses and tested over 7000 Daphnia magna genotypes for resistance to two strains of the bacterial pathogen Pasteuria ramosa. Results reveal the presence of strong epistasis between three closely linked resistance loci. One locus masks the expression of the other two, while these two interact to produce a single resistance phenotype. Changing a single allele on one of these interacting loci can reverse resistance against the tested parasites. Such a genetic mechanism is consistent with host and parasite specificity assumed by the Red Queen Theory. These results thus provide evidence for a fundamental assumption of this theory and provide a genetic basis for understanding the Red Queen dynamics in the Daphnia–Pasteuria system.     

Dissecting the genetic architecture of a stepwise infection process

How a host fights infection depends on an ordered sequence of steps, beginning with attempts to prevent a pathogen from establishing an infection, through to steps that mitigate a pathogen's control of host resources or minimize the damage caused during infection. Yet empirically characterizing the genetic basis of these steps remains challenging. Although each step is likely to have a unique genetic and environmental signature, and may therefore respond to selection in different ways, events that occur earlier in the infection process can mask or overwhelm the contributions of subsequent steps. In this study, we dissect the genetic architecture of a stepwise infection process using a quantitative trait locus (QTL) mapping approach. We control for variation at the first line of defence against a bacterial pathogen and expose downstream genetic variability related to the host's ability to mitigate the damage pathogens cause. In our model, the water‐flea Daphnia magna, we found a single major effect QTL, explaining 64% of the variance, that is linked to the host's ability to completely block pathogen entry by preventing their attachment to the host oesophagus; this is consistent with the detection of this locus in previous studies. In susceptible hosts allowing attachment, however, a further 23 QTLs, explaining between 5% and 16% of the variance, were mapped to traits related to the expression of disease. The general lack of pleiotropy and epistasis for traits related to the different stages of the infection process, together with the wide distribution of QTLs across the genome, highlights the modular nature of a host's defence portfolio, and the potential for each different step to evolve independently. We discuss how isolating the genetic basis of individual steps can help to resolve discussion over the genetic architecture of host resistance.     

Root herbivore identity matters in plant-mediated interactions between root and shoot herbivores

Plants are simultaneously attacked by a multitude of herbivores that affect plant responses and plant-mediated interactions in a variety of ways. So far, studies on indirect interactions between below- and aboveground herbivores have almost exclusively focused on interactions between only one root and one shoot herbivore species at the same time. Since these studies show a variety of outcomes, we test the hypothesis that root herbivore identity matters in below-/aboveground interactions. We studied the combined effects root-feeding nematodes (Pratylenchus penetrans) and wireworms (Agriotes lineatus larvae) on Plantago lanceolata and on the performance of aboveground phloem-feeding aphids (Myzus persicae) and chewing caterpillars (Chrysodeixis chalcites larvae). Since root herbivores may also affect resource availability and the microbial community in the rhizosphere, we examined resource utilization by soil microorganisms using BIOLOG EcoPlates™.

Wireworms decreased root biomass by 13%, but led to compensatory shoot growth. Nematodes and the aboveground herbivores did not affect the biomass of Plantago lanceolata. Feeding by C. chalcites larvae enhanced the concentration of aucubin in leaves, which might explain the high mortality of the caterpillars. Aphids and the belowground herbivores did not change iridoid glycoside levels in the leaves. However, the number of aphid offspring was reduced by 44% when nematodes had been added to the soil, whereas wireworms had no effect. We observed higher utilization of BIOLOG carbon sources by the soil microorganisms only in the presence of Pratylenchus penetrans. Our results suggest that the outcome of below–aboveground interactions highly depends on herbivore identity.     

Biological Control of Meloidogyne incognita by Paecilomyces lilacinus and Pasteuria penetrans

The root-knot nematode Meloidogyne incognita was controlled more effectively and yields of host plants were greater when Paecilomyces lilacinus and Pasteuria penetrans were applied together in field microplots than when either was applied alone. Yields of winter vetch from microplots inoculated with the nematode and with both organisms were not statistically different from yields from uninoculated control plots.     

Suppression Mechanisms of Meloidogyne arenaria Race 1 by Pasteuria penetrans

The biological control of Meloidogyne arenaria on peanut (Arachis hypogaea) by Pasteuria penetrans was evaluated using a six x six factorial experiment in field microplots over 2 years. The main factors were six inoculum levels of second-stage juveniles (J2) of M. arenaria race 1 (0, 40, 200, 1,000, 5,000, and 25,000 J2/microplot, except that the highest level was 20,000 J2/microplot in 1995) and six infestation levels of P. penetrans as percentages of J2 with endospores attached (0, 20, 40, 60, 80, and 100%). The results were similar in 1994 and 1995. Numbers of eggs per root system, J2 per 100 cm³ soil at harvest, root galls, and pod galls increased with increasing nematode inoculum levels and decreased with increasing P. penetrans infestation levels (P ≤ 0.05), except that there was no effect of P. penetrans infestation levels on J2 per 100 cm³ soil in 1994 (P> 0.05). There were no statistical interaction effects between the inoculum levels of J2 and the infestation levels of P. penetrans (P > 0.05). When the infestation level was increased by 10%, the number of eggs per root system, root galls, and pod galls decreased 7.8% to 9.4%, 7.0% to 8.5%, and 8.0% to 8.7% in 1994 and 1995, respectively, whereas J2 per 100 cm³ soil decreased 8.8% in 1995 (P ≤ 0.05). The initial infestation level of P. penetrans contributed 81% to 95% of the total suppression of pod galls, whereas the infection of J2 of the subsequent generations contributed only 5% to 19% suppression of pod galls. The major suppressive mechanism of M. arenaria race 1 by P. penetrans on peanut is the initial endospore infestation of J2 at planting.     

Temperature Effects on the Attachment of Pasteuria penetrans Endospores to Meloidogyne arenaria Race 1

Pasteuria penetrans is a gram positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. The attachment of the endospores to the cuticle of the nematodes is the first step in the life cycle of the bacterium and is essential for its reproduction. As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans on Meloidogyne arenaria race 1 were investigated. Preexposing second-stage juveniles (J2) of M. arenaria to approximately 30 °C in water before exposing them to endospores increased their receptivity to endospore attachment when compared to treating J2 at 25 °C or 35 °C. In tests with soil, highest attachment occurred when J2 were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating J2 in soil to sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. Incubating P. penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes as temperatures of incubation increased to 50 °C and higher.     

Effect of Temperature on Attachment,Development, and Interactions of Pasteuria penetrans on Meloidogyne incognita

The effect of temperature (10, 20, 25, 30, and 35 C) on attachment and development of Pasteuria penetrans on Meloidogyne arenaria race 1 was elevated in growth chambers. The greatest attachment rate of endospores of P. penetrans occurred on second-stage juveniles at 30 C. The bacterium developed more quickly within its host at 30 and 35 C than at 25 C or below. The development of the bacterium within the nematode female was divided into nine recognizable life stages, which ranged from early vegetative thalli to mature sporangia. Mature sporangium was the predominant life stage observed after 35, 40, 81, and 116 days at 35, 30, 25, and 20 C, respectively. The body width and length of M. arenaria females infected with P. penetrans were smaller initially than the same dimensions in uninfected females, but became considerably larger over time at 25, 30, and 35 C. This isolate of P. penetrans also parasitized and completed its life cycle in males of M. arenaria.     

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR‐based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria‐specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro‐organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.     

Persistence and Suppressiveness of Pasteuria penetrans to Meloidogyne arenaria Race

The long-term persistence and suppressiveness of Pasteuria penetrans against Meloidogyne arenaria race 1 were investigated in a formerly root-knot nematode suppressive site following 9 years of continuous cultivation of three treatments and 4 years of continuous peanut. The three treatments were two M. arenaria race 1 nonhost crops, bahiagrass (Paspalum notatum cv. Pensacola var. Tifton 9), rhizomal peanut (Arachis glabrata cv. Florigraze), and weed fallow. Two root-knot nematode susceptible weeds commonly observed in weed fallow plots were hairy indigo (Indigofera hirsuta) and alyce clover (Alysicarpus vaginalis). The percentage of J2 with endospores attached reached the highest level of 87% in 2000 in weed fallow, and 63% and 53% in 2002 in bahiagrass and rhizomal peanut, respectively. The percentage of endospore-filled females extracted from peanut roots grown in weed fallow plots increased from nondetectable in 1999 to 56% in 2002, whereas the percentages in bahiagrass and rhizomal peanut plots were 41% and 16%, respectively. Over 4 years, however, there was no strong evidence that endospores densities reached suppressive levels because peanut roots, pods, and pegs were heavily galled, and yields were suppressed. This might be attributed to the discovery of M. javanica infecting peanut in this field in early autumn 2001. A laboratory test confirmed that although the P. penetrans isolate specific to M. arenaria attached to M. javanica J2, no development occurred. In summary, P. penetrans increased on M. arenaria over a 4-year period, but apparently because of infection of M. javanica on peanut at the field site root-knot disease was not suppressed. This was confirmed by a suppressive soil test that showed a higher level of soil suppressiveness than occurred in the field (P ≤ 0.01).