To appropriately account for repeated measurements made over time, our analysis consisted of a linear mixed-effects model with binomial error, a random effect of block, and fixed effects of treatment , year , and symptom severity category . Next, we analyzed the rate at which PD reappeared in only severely pruned vines from category 3 in subsequent years using a survival analysis. Specifically, we used a Cox proportional hazards model with a fixed effect of plot .Accurate and time- or cost-efficient methods of diagnosing infected plants are important elements of a disease management program, both with respect to roguing to reduce pathogen spread , and the efficacy of pruning to clear plants of infection . Accurate diagnosis of PD in grapevines is complicated by quantitative and qualitative differences in symptoms among cultivars and other aspects of plant condition . Our results suggest that a well-trained observer can accurately diagnose PD based on visual symptoms, particularly for advanced cases of the disease. The small number of false positives in disease category 1 and 2 vines may have been due to misdiagnosis of other biotic or abiotic factors . Alternatively, false positives might indicate bacterial populations that are near the detection limit; conventional PCR has at least as low a detection threshold as other methods that rely on the presence of live bacterial cells . Regardless, although scouting based on visual symptoms clearly captured most cases of PD in the current study, some caution should be used when trying to diagnose early disease stages to ensure that vines are not needlessly removed. There is no cure for grapevines once infected with X. fastidiosa, plastic growers pots except for recovery that can occur in some overwintering vines .
The virulent nature of X. fastidiosa in grapevines, and the corresponding high mortality rate for early season infections, increases the potential value of any cultural practices that can cure vines of infection. Moreover, new vines replanted into established vineyards generally take longer to develop compared to vines planted in newly developed vineyards, potentially due to vine-to-vine competition for resources that limits growth of replacement vines. As a result, vines replanted in mature vineyards may never reach full productivity . Thus, management practices that speed the regeneration of healthy, fully developed, and productive vines may reduce the economic loss caused by PD . A multinomial logistic regression showed significant differences in the relative frequency of different grapevine growth outcomes between the two restoration methods . Chip-budded vines showed significantly lower frequency of strong growth and significantly higher frequencies of vines with developing growth and, especially, of no growth . Nearly 30% of chip-budded vines showed no growth in the following season, compared to 0% of vines on which established shoots were trained. These results indicate that training newly produced shoots from the remaining section of the scion was more likely to result in positive regrowth outcomes. As a result, of the two methods we evaluated, training of shoots that emerge from the scion of a severely pruned trunk is recommended for restoring growth. However, it is important to note that the current study did not estimate the amount of time required for severely pruned vines to return to full productivity. Moreover, the study did not include mature vines, in which growth responses may differ from young vines.
Additional studies may be needed to quantify vine yield, and perhaps fruit quality, in severely pruned vines over multiple seasons. The usefulness of pruning for disease management depends on its ability to clear plants of pathogen infection . A comparison of symptom prevalence among severely pruned and control vines from different disease severity categories showed significant effects of the number of years after pruning , pruning treatment , and initial disease symptom category . The analysis also showed significant interactions between year and treatment and between treatment and symptom category , a non-significant interaction between year and symptom category , and a marginally significant three-way interaction . Overall, more vines had symptoms in the second year compared to the first , and there was a higher prevalence of returning symptom in vines from higher initial disease categories . Severe pruning showed an apparent benefit to reducing symptoms of PD after the first year, but this effect weakened substantially by the second year, with no differences for category 1 or 3 vines, and a slightly lower disease prevalence for severely pruned category 2 vines . A survival analysis of severely pruned category 3 vines showed a significant difference in the rate of symptom return among plots . All vines in plots 1 to 3 had symptoms by autumn 2000, two years after pruning . In plots 4 and 5, more than 80% of vines showed symptoms after three years. Only plot 6 showed markedly lower disease prevalence; in plot 6, ~70% and 50% of severely pruned category 3 vines showed no symptoms after two and four years, respectively, versus ~36% of control vines overall, after two years. It is important to note that at the time of this study, disease pressure may not fully explain the return of symptoms in severely pruned vines.
Surveys conducted during the first two years of the study throughout the entirety of the six research blocks showed that the prevalence of PD in control vines actually declined slightly from the first to the second year , but not due to an increase in replanting efforts or vine death , Rather, this decline in prevalence likely reflects overwinter recovery of mild cases of the disease . Thus, the observed return of symptoms in most severely pruned vines does not appear to be explained by reinfection with X. fastidiosa after clearing of infection during the severe-pruning process. Our results indicate that the apparent effectiveness of severe pruning depended on the initial disease severity, and the effectiveness weakened over time. This suggests at least two constraints exist regarding the general utility of pruning as a PD management tool. First, severe pruning does not appear to be useful for mild cases of PD, as many of those same vines would recover from the infection over the winter . Second, there appears to be little value in pruning severely diseased vines; the high frequency of symptom return within a few years indicates that even severe pruning does not clear most vines of X. fastidiosa infection. That leaves a statistically significant window with respect to intermediate severity cases, which may benefit from severe pruning. The apparent benefit for this category of diseased vines would stem from infections that are not so localized that they are highly susceptible to natural recovery over the winter, but also not fully systemic such that the infection has developed below the pruning point . Reliable identification of this narrow class of diseased vines may require substantial experience with PD scouting, detailed record keeping, and an appreciation for variability in symptoms or infection dynamics based on grapevine cultivar and environmental conditions . Research in other bacterial plant pathosystems has evaluated the potential benefit of pruning and whether pruning extent is related to its effectiveness at clearing hosts of infection . A study of the citrus disease huanglongbing, blueberry in pot associated with infection by Candidatus Liberibacter spp., evaluated two levels of pruning severity, neither of which showed promise as a disease management tool . In this pathosystem, it is plausible that a very protracted incubation period may undermine the effectiveness of pruning, because by the time the first symptoms are visible, the infection may have already moved throughout much of the tree. Collectively, our results are more similar to a study of citrus variegated chlorosis . In this study, the presence of X. fastidiosa in plant tissues at different distances from symptomatic leaves was determined for varying levels of disease severity. X. fastidiosa was more widely distributed in trees with severe disease symptoms compared to those with early stage foliar symptoms. Although ColettaFilho et al. did not test whether pruning at various distances proximal to symptomatic leaves would eliminate X. fastidiosa infections, the current recommendation is to prune citrus material if early symptoms are present, and to not prune plants with severe disease symptoms . Citrus plant age is also an important consideration; Coletta-Filho and de Souza recommend that symptomatic citrus trees up to three-years-old be removed rather than pruned, whereas trees four-years-old or older should be pruned. We did not examine vine age as a factor in this study, but the biology of citrus and grape differ in terms of the overwinter recovery that can occur in grape and the apparently slower movement of X. fastidiosa in citrus compared to grape. Anecdotally, the two most mature plots in our study showed the most rapid return of disease, and the youngest plot showed the slowest return.
More studies of the effect of vine age are needed before concluding that interactive effects of plant age and pruning differ between the PD and citrus variegated chlorosis pathosystems.Most bee species are considered mass provisioners—i.e., they build a brood cell into which they pack a mixture of pollen and nectar, deposit an egg on the pollen provision, and seal off the brood cell while the offspring develops. This brood cell is left sealed until the fully developed bee emerges by breaking through the cell cap. While the mother bee creates the pollen provision for the developing bee, numerous other creatures may enter the brood cell. Organisms found in bee brood cells include—but are surely not limited to—nematodes, mites, springtails, bacteria, and fungi. The bee brood cell can therefore be considered a miniature ecosystem, and how the interactions occurring within these tiny ecosystems affect bee health is a fascinating question. High-throughput sequencing has allowed for detailed surveys of the diversity of the microbes that inhabit pollen provisions. Early next-generation sequencing surveys of pollen provisions suggested that many bacteria found in pollen provisions may be acquired from flowers. The observation that the same bacteria inhabit flowers, pollen provisions, and bee guts was subsequently verified. Further studies then linked foraging to microbial transmission, which is more apparent when characterizing a network of plants, multiple bee species, and bacteria than when studying a single population of bees. When looking at multiple populations of one species across habitats, pollen usage and fungi co-vary more than pollen usage and bacteria. The consensus arising from these studies is that flowers serve as transmission hubs for pollen-associated microbes, but the characteristics of pollen provisions may determine which microbes thrive there. The genomes of pollen-associated lactobacilli contain genes involved in osmotic stress tolerance, detoxification of metals and other toxicants, and pollen wall degradation. That these microbes exhibit genomic adaptations for rapid growth in nutrient-rich environments suggests that they likely ferment sugars found in pollen provisions and may exclude spoilage organisms, as their close relatives do in sourdough bread dough. Experimental evidence for a nutritional role of pollen-borne microbes is also mounting. Isotopic signatures of diet suggest that bee larvae from a diversity of bee species are not truly herbivores as one would expect, but instead exhibit omnivorous or even carnivorous traits. This finding suggests that bee larvae are consuming microbes in their pollen provisions. Feeding bee larvae different ratios of sterilized to normal pollen leads to differences in growth rates and survival, again suggesting that larvae consume pollen-borne microbes. Similarly, whether microbes were present or absent in pollen had a greater influence on larval development compared to whether the pollen was collected by con-specific or different bee species for larvae of the specialist blueberry pollinator Osmia ribifloris. Altogether, these studies are beginning to illustrate the importance of microbes in the pollen provisions of wild and solitary bees for larval health. One open question in the study of the microbiome of the pollination landscape is how diet breadth affects exposure to and acquisition of microbes. Across the bee phylogeny, there is a diversity of diet breadths, with some bees visiting a broad diversity of plant species and others visiting a limited number of plants , or even a single plant , with gradations in between these groups. As specialist bees visit fewer plants, they may acquire a distinct microbial community compared to generalist bees. Conversely, if specialists interact with the same plants as generalists do, both classes of bees may be exposed to the same microbes. The microhabitats of specialist and generalist bee pollen provisions may filter different microbes based on pollen and nectar chemistry, altering microbial composition as has been found with nectar microbial communities.