Runsick, B., County Extension Agent - Agriculture, University of Arkansas Division of Agriculture Cooperative Extension Service

ABSTRACT

The effects of sting nematodes (Belonolaimus longicaudatus) on seven different cultivars of forage-type tall fescue (Festuca arundinacea Schreb.) were evaluated in the greenhouse.  Cultivars infected with the toxic endophyte, Neotyphodium coenophialum, novel endophyte cultivars, and endophyte-free cultivars were included in the study.  Both the suitability of each cultivar as a host for B. longicaudatus and the effects of the nematode on root weights were evaluated.  In addition to the greenhouse studies, a survey of five northern Arkansas counties was conducted to determine the presence of plant-parasitic nematodes in established tall fescue pastures.  Fifty-four samples from fescue pastures in Baxter, Benton, Sharp, Madison, and Fulton counties  contained stubby-root (Paratrichodorus spp.), stunt (Tylenchorhynchus spp.), dagger (Xiphinema spp.), lance (Hoplolaimus spp.), root-knot (Meloidogyne spp.), pin (Paratylenchus spp.), and Tylenchus spp.  Population densities were low in all pastures likely because of low soil moisture conditions at the time of sampling.

Introduction

According to the University of Arkansas Cooperative Extension Service, tall fescue (Festuca arundinacea L.) is one of the most common forage pasture and hay grasses and total over 2 million acres, statewide (Jennings, Beck, & West, 2008).  Tolerance to environmental stresses can, in part, be attributed to the presence of the endophyte, Neotyphodium coenophialum (West, 1994; Malinowski & Belesky, 2000; Malinowski et al., 2005).  Fescue is a host for several nematode species, worldwide including cyst nematodes (Heterodera spp.), root-knot nematodes (Meloidogyne spp.), seed gall nematodes (Anguina spp.), lesion nematodes (Pratylenchus spp.), and spiral nematodes (Helicotylenchus spp.) (Thies et al., 1995).  Some, although not all, populations of the sting nematode (Belonolaimus spp.) also may parasitize fescue (Robbins & Barker, 1973).

The presence of N. coenophialum may confer resistance to certain nematode species (Elmi et al., 2000; Gwinn & Bernard, 1993).  Although the mechanism is not completely understood, it appears that thickening of endodermal cell walls in endophyte infected fescue plants may play a role in resistance to nematodes (Gwinn & Bernard, 1993). This resistance may be derived from plant responses to the endophyte or from the endophyte itself.  Roberts, et al. (1992) found that when Meloidogyne sp. infected both endophyte-infected and endophyte free ‘Kentucky 31’ plants, the endophyte-infected plants produced higher levels of chitinase.  Hypersensitivity in response to nematode challenge and compounds, such as phytoalexins and pathogenesis-related proteins also play a role resistance to nematodes (Lindgren, Smith, & Jakobek, 1992).  Root extracts of endophyte-infected fescue include various ergot alkaloids (ergovaline, ergotamine, a-ergocryptine, ergonovine), and loline alkaloids which can alter chemotaxis and inhibit orientation to roots by Pratylenchus scribneri whereas root extracts of endophyte-free fescue may act as attractants to the nematode (Bacetty, 2009).  In this study, the alkaloid, ergovaline, also showed nematicidal effects at both high and low concentrations.

This apparent contribution to enhanced resistance to nematodes by N. coenophialum in tall fescue is also dependent upon both the strain of the endophyte that is present and the fescue cultivar.  Timper, et al., (2004) found that after 8 weeks the number of Pratylenchus spp. in roots of the fescue cultivars ‘Jesup’ and ‘Georgia 5’ infected with the N. coenophialum non-ergot strain, AR542 was no different than those endophyte free (E-) controls.  The non-ergot strain, AR584, however, conferred resistance to the nematode in Georgia 5’, but not in ‘Jesup’, although the level of resistance was less than that with the endemic endophyte.  The basis for these differences in conferred resistance among different cultivars and strains of N. coenophialum is unknown.

Materials and Methods

Fescue cultivars were selected based on endophyte status, including one “wild type” endophyte-infected cultivar, three novel-endophyte-infected cultivars, and three endophyte-free cultivars.  The endophyte-infected cultivar used was ‘Kentucky 31’.  Novel endophyte cultivars included ’Jesup MaxQ’, ‘BarOptima PLUSE34’, and ‘Duramax GOLD’.  The endophyte-free cultivars were ‘AU Triumph’, ‘Barolex’ and ’Duramax’.  Paired comparisons (six replications) with and without nematodes were used to compare the fescue cultivars for their suitability as hosts for B. longicaudatus.  Twenty-five seeds of the appropriate cultivar were sown on the surface of 4-inch (10.2 cm) diameter clay pots containing 300 cm³ (18.31 in³) of a steam pasteurized fine sand to facilitate sting nematode reproduction (Robbins & Barker, 1971; Robbins & Barker, 1974).  Pots were inoculated with 1 male and 5 female B. longicaudatus that had been hand-picked from a culture maintained on bermudagrass (Cynadon dactylon L.).  Fescue pots were maintained in a greenhouse at 29.4°C – 32.2°C (85°F – 90°F).  Studies by Bekal and Becker (2000) indicated that population densities of B. longicaudatus in bermudagrass turf peaked when soil temperatures were between 25°–30°C (77°F – 86°F). The plants were watered three times daily and fertilized with Osmocote™ 14-14-14 at a rate of 1.0 g (0.035 ounces) per pot on days 38, 55, 77, and 91.  Plants remained in a vegetative growth stage for the duration of the experiment.

To allow ample time for root growth after planting, the fescue plants were grown for 34 days before being inoculated with 1 male and 5 female B. longicaudatus.  Small trenches that reached into the root zone were dug in each pot, and the nematodes, in 10 ml (0.61 in³) water, were poured into the trench and covered.  Plants were allowed to grow for 64 days after inoculation to allow time for B. longicaudatus to complete 2 generations of its life cycle.  At the conclusion of the experiment, the roots and soil were removed from each pot and separated by gently shaking.  The soil was then assayed using elutriation and centrifugal flotation as described earlier for survey samples.  The roots were then collected and dried [60ºC (140 ºF) for 48 hr] and weighed. 

Results and Discussion

Nematode numbers increased on all each of the cultivars tested across all cultivars. This level of reproduction indicates that all of the cultivars  were excellent hosts for B. longicaudatus using the criteria established by Robbins & Barker (1973) who considered a plant species to be an excellent host if reproduction was greater than eightfold. That is, any increase of more than 48 individuals (6 original individuals multiplied by 8) would be considered a high enough level of reproduction to consider the cultivar a good host. Nematode populations in individual pots ranged from 17 to more than 5,000 (data not shown).

The presence or absence of the endophyte did not affect the reproduction of B. longicaudatus (Table 1).  The sting nematode feeds exclusively as an ectoparasite.  Kimmons et al. (1990) reported that reproductive rates of the ectoparasitic nematode, Helicotylenchus pseudorobustus, were not significantly different in endophyte-infected and endophyte-free fescue cultivars, whereas the reproduction of the endoparasitic nematodes, Pratylenchus scribneri and Meloidogyne marylandi were adversely affected by the endophyte.  They speculated that endoparasitic nematodes may either be exposed to inhibitory compounds for longer periods of time than ectoparasites, or they may be subject to exposure to different compounds due to their physical presence inside host roots.   It is also possible that while the endophytes studied in this trial conferred some degree of resistance to the nematode, the sensitivity of our trial was not adequate to detect subtle differences that may have occurred.  At any rate, very little effect was detected regardless of cultivar-endophyte combination.  It is unclear why the presence of the nematode tended to increase root dry weight compared with plants that were nematode-free in some cultivars.  Compensatory root growth in response to nematode infection has been reported with both potato (Smit & Vamerali, 1998) and cotton (Ma, et al., 2012).  It is unlikely, however, that this situation would have occurred under field conditions.

Fescue cultivar and endophyte status a	# of sting nematodes 64 days after inoculation b	Average Dry Root Massb
‘Kentucky 31’ (E+)           control           inoculated	- 1,765 A	7.20g (0.016 lbs) 8.55g (0.019 lbs)
‘Jesup MaxQ’ (NE+)           control           inoculated	- 1,407 A	5.25g (0.012 lbs)* 7.78g (0.017 lbs)
‘BarOptima PLUSE34’ (NE+)           control           inoculated	- 1,335 A	9.02g (0.020 lbs) 10.87g (0.024 lbs)
‘Duramax GOLD’ (NE+)           control           inoculated	- 1,338 A	7.53g (0.017 lbs)* 10.26g (0.023 lbs)
‘Duramax’ (E-)           control           inoculated	- 641 A	3.62g (0.008 lbs)* 9.25g (0.020 lbs)
‘AU Triumph’ (E-)          control          inoculated	- 1,740 A	6.52g (0.014 lbs) 7.42g (0.016 lbs)
‘Barolex’ (E-)           control           inoculated	- 1,672 A	4.70g (0.010 lbs)* 8.62g (0.019 lbs)

^aE+, NE+, and E- indicate endophyte infected, novel endophyte, and endophyte free fescue cultivars, respectively.

^bNematode means followed by the same letter do not differ at P = 0.05 according to LSD.

^*Significant difference at (P < 0.05)

Table 1.  Effects of tall fescue (Festuca arundinacea) cultivar and endophyte status on reproduction of sting nematodes (Belonolaimus longicaudatus) and plant root mass.

Conclusions and Future Research

Endophyte status did not appear to influence the reproduction of B. longicaudatus in this study.  Further research should be conducted using inoculum from different populations, as previous research has indicated varied host susceptibility of plants inoculated with B. longicaudatus derived from different locations (Robbins & Barker, 1973).  Additionally, further research should be conducted with plants grown under varying environmental conditions, and a greater number of replications may be included in the experiment. The presence of sting nematodes had a significant positive effect (P <  0.05) on fescue cultivar dry root weights.  This was likely an artifact of plants that were grown in a greenhouse environment for a relatively short period of time, and although compensatory root growth may have been a factor where environmental stresses were minimal, sting nematode infection in the field would not likely be an asset to the fescue plant.  

Note to Readers:

It is understood that there is a major flaw in this research relating to the endophyte status. It was assumed, perhaps inappropriately, that the E+ and NE+ cultivars did contain the endophyte and its associated alkaloids and the E- did not, without having actually tested for it. This was due, in part, mostly to the lack of foresight by the author, as well as time and financial constraints.  This should be addressed in any further research conducted regarding this host/pathogen interaction.

Acknowledgements

Special thanks to assisting county agriculture agents, Robert Seay, Mark Keaton, Darrin Henderson, and Joe Moore for their work with the survey sampling, and to University of Arkansas Nematode Diagnostic Lab staff, Ronnie Bateman, Margie Miller, Tammy Woodruff, and Cathy Howard for their work with nematode counts, extraction, and greenhouse work.  Additionally, thanks is given to Dr. Don Edgar for statistical analysis assistance and Dr. Terry Kirkpatrick for oversight and guidance given in this research.

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