Michael W. Hotchkiss

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strains virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.

Suppressive effects of metabolites from Photorhabdus and Xenorhabdus spp. on phytopathogens of peach and pecan.

Abstract Our objective was to determine the suppressive abilities of bacterial metabolites derived from Xenorhabdus and Photorhabdus spp. on Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, and Fusicladosporium effusum, which are fungal or oomycete pathogens of pecan, and Monilinia fructicola, a fungal pathogen of peach. In the first set of in vitro assays, when metabolites were compared based on initial bacterial cell count, X. bovienii (SN) metabolites generally exhibited the greatest suppression of phytopathogens and Xenorhabdus sp. (355) the least with Photorhabdus luminescens (Hb) and Xenorhabdus nematophila (All) being intermediate. In a second set of in vitro assays, in which metabolites were compared at 50 mg per ml acetone, P. luminescens (VS) exhibited greater suppression than P. luminescens (Hb), Photorhabdus sp. (MX4), X. bovienii (SN), and Xenorhabdus sp. (3 – 8b). In in vivo tests, 6 or 12% dilutions of X. bovienii (SN) or P. luminescens (Hb) metabolites caused 90 – 100% suppression of P. cactorum lesions on pecan leaves with only slight phytotoxicity. No phytotoxic effects were observed in detached peach leaves at dilutions up to 25%. Metabolite treatments, derived from X. bovienii (SN) and P. luminescens (Hb), were also tested for suppression of F. effusum sporulation in detached pecan shoots. Reductions in sporulation caused by bacterial metabolites were similar to those following treatment with two chemical fungicides, dodine and fenbuconazole; a third chemical triphenyltin hydroxide had no effect. Further research is warranted to determine if fungal or oomycete incited diseases in pecan and peach can be controlled with metabolites of Xenorhabdus spp. and Photorhabdus spp.

Comparative Impact of Artificial Selection for Fungicide Resistance on Beauveria bassiana and Metarhizium brunneum

ABSTRACT Hypocreales fungi such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium brunneum Petch can be negatively affected by fungicides thereby reducing their biocontrol potential. In a previous study, we demonstrated enhanced fungicide resistance in B. bassiana through artificial selection. However, it is not clear if the enhanced resistance was because of improved germination, vegetative growth, or both. Additionally, the enhanced fungicide resistance has only been demonstrated in B. bassiana, and therefore it is of interest to investigate the potential to enhance resistance in other fungi. Thus, the objectives in this study were to determine the potential to enhance fungicide resistance in M. brunneum through artificial selection, and investigate if selection is based on germination, vegetative growth, or both in B. bassiana and M. brunneum. Selection for resistance to fenbuconazole, and triphenyltin hydroxide was assessed through inhibition evaluations on solid media, and germination and mycelial growth in liquid media. Increased resistance after selection was observed for all fungicide-fungus combinations on solid and or liquid media. Selection resulted in increased resistance to fenbuconazole in both fungi in solid and liquid media; in liquid culture fungicide resistance in B. bassiana was manifested by increased germination and mycelial growth, whereas in M. brunneum fungicide resistance concerned only mycelial growth. Selection for resistance to triphenyltin hydroxide varied in the different media. For B. bassiana, triphenyltin hydroxide resistance was enhanced on solid media but not in liquid, whereas enhanced resistance of M. brunneum was detected in both media. Fungicide sensitivity and selection potential differs based on the medium and fungal species. Selection for fungicide resistance, had negative effects on other beneficial traits when fungicide pressure was removed, for example, some selected populations showed decreased germination or growth, relative to their nonselected control populations. Additionally, reduced virulence to the greater wax moth, Galleria mellonella (L.), was observed in all fungal populations that were exposed to fungicide resistance regimes. We conclude that it is possible to use genetic selection to enhance fungicide resistance in B. bassiana and M. brunneum, but before use the resulting populations should be screened for inadvertent negative impacts on beneficial traits.

The Effect of Sample Height on Spray Coverage in Mature Pecan Trees

Pecan scab (caused by Fusicladium effusum) is the most damaging disease of pecan in the southeastern United States. Large air-blast sprayers for orchards are used to apply fungicide to control the disease but little quantitative information exists on the spray coverage achieved in the canopy of these trees. A series of experiments using water-sensitive spray cards to record spray coverage (percent area) at different heights and locations up to 15 m in the canopy of pecan trees showed a significantly greater percentage of card area covered at the lowest sample height when compared with the highest sample height. At the lowest height (5 m), spray coverage on individual cards ranged from 4.7 to 73.5% and, at the highest sample height (15 m), spray coverage ranged from 0.02 to 9.5%. In general, there was little significant difference in spray card coverage up to at least 10 m but, at 12.5 and 15 m, there was significantly less spray coverage compared with the coverage at 5 m. Regression analysis indicated a consistent linear relationship between sample height in the tree and the percent area covered. When spray cards were positioned at different heights without possible interference from pecan limbs and foliage, similar effects of sample height on spray coverage were noted. Wind speed measurements showed that air movement declined rapidly with distance from the sprayer fan. Whereas, at 2 m from the fan, wind speeds were approximately 26 m s-1, by 10 m, speeds had declined to 2 to 4 m s-1. At distances >12 m, wind speed was approaching ambient air movement of about 1 to 3 m s-1. Although aerial application resulted in numerically greater spray coverage at sample heights >10 m, it was not significant even though a weak linear relationship (R2 = 0.21 to 0.25) suggested an effect of height. Characterizing and understanding pesticide spray coverage in pecan will allow us to discern limits imposed by existing technology, and provide the basis for improving spray application methods (or tree management) for more efficacious disease control.

The distribution of peach scab lesions on the surface of diseased peaches

The aim of the study was to quantify peach scab (Fusicladosporium carpophilum) lesion distribution relative to the point of maximum lesion number on the fruit surface, the relationship between lesion count and distance from the point of maximum lesion density, and establish whether the distribution of lesions was consistent with a splash dispersed pathogen, and to assess the effect of lesion number on fruit size. Fruit of four cultivars, Jerseyqueen, Jefferson, BY07-6428r and Dixiland were collected and the fruit (assumed spherical) sliced taking three horizontal planes across the axis from the point of maximum disease, such that each horizontal zone (Z1-Z4) had the same vertical height, and thus equal surface areas. Lesion counts were analysed using general linear modeling with a Poisson distribution and a log-link function. Zones on the fruit had different numbers of lesions (P < 0.0001), with most lesions found on Z1. Cvs differed in the number of lesions per fruit (P = 0.0042–<0.0001). An analysis of covariance showed that although fruit size varied among most cvs (P = 0.1614–<0.0001), the number of lesions on a fruit did not affect fruit size (P = 0.5654). Measurements of the point of maximum disease relative to the peduncle-flower scar axis of the fruit suggest that fruit are not always held upright when infection occurs, such that up to 40% of fruit showed maximum infection at an angle >90° to the peduncle. This pattern of disease is consistent with observations of the splash-borne nature of conidia, with the most exposed, easily wetted, uppermost portion of fruit developing most disease.

Severity of Scab and its Effects on Fruit Weight in Mechanically Hedge-Pruned and Topped Pecan Trees

Scab is the most damaging disease of pecan in the southeastern United States. Pecan trees can attain 44 m in height, so managing disease in the upper canopy is a problem. Fungicide is ordinarily applied using ground-based air-blast sprayers. Although mechanical hedge-pruning and topping of pecan is done for several reasons, improved management of scab is an important reason in the humid, wet Southeast. Resulting shoot growth on cut limbs of susceptible cultivars could lead to more severe scab. In three experiments over three years, we explored the effect of hedge-pruning trees to ∼12 to 14 m compared with non-hedge-pruned trees. All trees received fungicide treatments (air-blast sprays and ≤3 aerial applications). Hedge-pruning either had no effect, or increased or decreased scab severity only slightly on leaflets, immature, or mature fruit (a -9.95 to +14.63% difference in scab severity compared with the control). However, height in the canopy invariably had a large and significant effect on scab severity, and amounted to a 0.05 to 73.77% difference in severity between the lowest and highest sample in the canopy. Fruit weight depended on sample height, with fruit most often weighing less when collected at greater sample heights. A robust relationship between fruit weight and scab severity was found at the highest sample heights where scab was also most often severe (R2 = 0.21 to 0.67, P < 0.0001). Hedge-pruning and topping pecan tree canopies to manage tree size will enable better fungicide coverage, reducing risk of a scab epidemic as more of the canopy is assured efficacious fungicide spray coverage.

Population Genetic Structure of Venturia effusa, Cause of Pecan Scab, in the Southeastern United States

Venturia effusa is the most important pathogen of pecan in the southeastern United States. Little information exists on the population biology and genetic diversity of the pathogen. A hierarchical sampling of 784 isolates from 63 trees in 11 pecan orchards in the southeastern United States were screened against a set of 30 previously characterized microsatellite markers. Populations were collected from Georgia (n = 2), Florida (n = 1), Alabama (n = 2), Mississippi (n = 1), Louisiana (n = 1), Illinois (n = 1), Oklahoma (n = 1), Texas (n = 1), and Kansas (n = 1). Clonality was low in all orchard populations (≤10.1% of isolates), and there were consistently high levels of genotypic diversity (Shannon-Weiners index = 3.49 to 4.59) and gene diversity (Neis measure = 0.513 to 0.713). Analysis of molecular variance showed that, although 81% of genetic diversity occurred at the scale of the individual tree, 16% occurred between orchards and only 3% between trees within orchards. All populations could be differentiated from each other (P = 0.01), and various cluster analyses indicated that some populations were more closely related compared with other pairs of populations. This is indicative of some limited population differentiation in V. effusa in the southeastern United States. Bayesian and nearest-neighbor methods suggested eight clusters, with orchards from Georgia and Florida being grouped together. A minimum spanning tree of all 784 isolates also indicated some isolate identification with source population. Linkage disequilibrium was detected in all but one population (Kansas), although 8 of the 11 populations had <20% of loci at disequilibrium. A Mantel test demonstrated a relationship between physical and genetic distance between populations (Z = 11.9, r = 0.559, P = 0.001). None of the populations were at mutation-drift equilibrium. All but 3 of the 11 populations had a deficiency of gene diversity compared with that expected at mutation-drift equilibrium (indicating population expansion); the remaining populations had an excess of gene diversity compared with that expected at mutation-drift equilibrium (indicating a recent bottleneck). These observations are consistent with the known history of pecan and pecan scab, which is that V. effusa became an issue on cultivated pecan in the last approximately 120 years (recent population expansion). Recently reported mating type genes and the sexual stage of this fungus may help explain the observed population characteristics, which bear a strong resemblance to those of other well-characterized sexually reproducing ascomycete pathogens.

Scab Susceptibility of a Provenance Collection of Pecan in Three Different Seasons in the Southeastern United States

Pecan scab (caused by Fusicladium effusum) is the most economically destructive disease of pecan in the Southeast United States. Wet, humid conditions typical of the Southeast are known to provide conditions conducive to epidemics. A provenance collection of pecan from 19 locations representing the native range of the tree is located in Byron, Georgia, and was assessed for pecan scab severity in 1998, 2013, and 2014. There were significant differences among the 19 provenances (F = 5.6 to 62.5, P < 0.0001). Provenances from wetter locations (generally north of Texas) had the greatest proportion of scab resistant trees, while provenances from the drier southern areas (Texas and Mexico) tended to be the most susceptible to scab. The association with rainfall was borne out by correlation analysis (r = -0.625 to -0.823 [P < 0.0001 to 0.004]). Other factors consistently associated with scab severity included leaflet tilt and droop angle (r = -0.533 to -0.883 [P < 0.0001 to 0.02]). Multiple regression analysis demonstrated that leaflet droop angle was a particularly good predictor of provenance susceptibility. Leaflet characteristics vary with provenance location, and whether there is a direct relationship between scab severity and leaflet characteristics is not established. Estimates of heritability were not entirely consistent among years, but different methods were used to assess scab severity in 1998 (a 1 to 5 category scale) compared with 2013 and 2014 (the percent ratio scale). Despite using different methods, there was generally good agreement among years in regard to severity of disease on individual trees. In conclusion, trees from more northern populations (in areas with greater annual rainfall) are most likely to provide valuable and diverse sources of resistance to scab. The provenance collection contains a range of scab-resistant genotypes from diverse locations that can contribute to genetic improvement regarding scab resistance.

A comparison of organic fungicides: alternatives for reducing scab on pecan

In the southeastern USA, the most widespread and damaging disease of pecan is scab, caused by Venturia effusa. Although scab can be controlled using conventional chemical methods, organic pecans that attract a premium price mandate the use of organic fungicides. Also, organic production is an environmentally sustainable method. However, where susceptible pecan cultivars are grown, there are limited options for organic management of scab. We conducted experiments to compare organic fungicides to control scab on the susceptible cv. Desirable in 2011, 2012, 2014, 2015, and 2016. The alternatives compared included Bordeaux mixture, compost tea, sodium bicarbonate, Bacillus subtilis, sulfur, cuprous oxide, and extract of the Giant Knotweed (Reynoutria sachalinensis). Rainfall and scab severity differed between seasons. There was consistently low severity on foliage, with little or no difference between treatments. Similarly at the time of the first fruit assessment, the severity was low and the differences in severity small and inconsistent between seasons and treatments. However, by the time of the second fruit assessment, severity of scab had increased and consistent differences among treatments existed (except in the drought year of 2011, when scab severities were very low and similar to the control). In all other years, the control treatment had significantly more severe scab compared to some (2012 and 2014) or all other treatments (2015 and 2016). Extract of the Giant Knotweed as a fungicide was included in 2012, 2014, 2015, and 2016, and fruit on those trees had less severe scab in all years compared to that on fruit of the control trees. In three seasons (2012, 2015, and 2016), applications of Bordeaux mixture resulted in a reduction in scab severity. Compost tea, Sodium bicarbonate, B. subtilis, sulfur, and cuprous oxide significantly reduced scab compared to the control in one or two seasons, but were not consistent among seasons, and were never more efficacious compared to the extract of the Giant Knotweed. Extract of the Giant Knotweed and Bordeaux mixture appear to offer the greatest potential as organic approaches for managing scab in pecan. However, wherever possible, planting of scab resistant cultivars should be considered as a first line of defense.