Journal of Soil Science and Plant Nutrition, 2017, 17 (1), 91-98 SHORT COMMUNICATION

Incidence of bonariensis (Coleoptera: ) in ryegrass pastures from southern Chile. New background

Leonardo Parra1,2,3*, Manuel Chacón-Fuentes2,3, Marcelo Lizama2,3 and Andrés Quiroz2,3

1Scientific and Technological Bioresource Nucleus, BIOREN-UFRO, Universidad de La Frontera, Temuco, Chile. 2Laboratorio de Química Ecológica, Departamento de Ciencias Químicas y Recursos Naturales, Uni- versidad de La Frontera, Temuco, Chile. 3Centro de Investigación Biotecnológica Aplicada al Medio Ambiente (CIBAMA), Universidad de La Frontera, Av. Francisco Salazar 01145, Temuco, Chile. *Correponding author: [email protected]

Abstract

The Argentine stem , Listronotus bonariensis, is an important pest in pastures, especially ryegrass (Lo- lium sp.), compromising grassland persistence and productivity. Both, adult and specially larvae cause severe damage, action that generate the death of the tiller and reducing forage production. Chemical control has lim- ited effectiveness in adult’s and negative effects on the environmental and toxicity to pollinators when applied close to the flowering period is observed. Therefore, the main currently strategy is based on the use of cultivars of Lolium endophyte fungus-infected with Neothypodium genus. However, over a decade that there are no reports on the presence of this weevil in southern Chile. Hence, the aim of this research was report the actual incidence of L. bonariensis in ryegrass pastures. The results of two years of prospecting showed that this weevil is currently causing damage to the ryegrass pastures indicating that the effectiveness of infected with the fungus must be evaluated. Keywords: Damage, distribution, immature stage, endophyte, weevil

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1. Introduction

The Argentine stem weevil Listronotus bonariensis Neothypodium genus and they are resistant to feed- (Coleoptera: Curculionidae), a native species of South ing larvae and adults of L. bonariensis (Spiering et America, is an important pest in pastures, especially al., 2005; Moore et al., 2015). In general, Neotypho- ryegrass (Lolium sp.), wheat and barley, because their dium endophytes have been used as an effective tool attack occurs in two stages of development. Weevil for controlling grassland pest due offer protection larvae bore the stem and crowns compromising pas- against porina larvae Wiseana cervinata (Lepidop- ture persistence and productivity, while the adult stage tera: Hepialidae), African black Heteronychus can reduce seedling establishment by defoliation and arator (Coleoptera: Scarabaeidae) and L. bonariensis cutting of the stems (Baker and Addison 2006; Barrat (Jensen and Popay, 2004; Vidal and Jaber, 2015). This et al., 2016). In New Zealand this insect causes yield topic has been studied extensively in New Zealand losses in a range between 5 and 30% in L. perenne during the last decade by Popay et al., (2003). In this and economic losses that reach the U.S. $ 138 million/ regard, an updated study in laboratory and field condi- year (Prestidge et al., 1991). In the same country, the tions, conducted with larvae of Costelytra zealandica densities of L. bonariensis reach between 130,000- (Coleoptera: Scarabaeidae), one of the largest subter- 400,000/ha (Barratt et al., 2007). In Chile, the first re- ranean pests in pastures of New Zealand, determined cord on the incidence of L. bonariensis was reported a negative relationship between the content of lolines by Norambuena and Gerding (1985) on wheat and in roots of fescue and consumption and weight gain barley in the south of the country, detecting 27.9% by larvae (Patchett et al., 2011). This effect would be and 7.1% attacked axes, respectively. Subsequently, detrimental to the insect, and it would be induced by Cisternas (2001) indicated that in Osorno (Región de the same larva, which feed on the root triggering a Los Lagos) were determined losses of 44.4% dry mat- defensive process of the plant, which would transport ter production in L. multiflorum (ryegrass biannual) the alkaloid from the crown to the root (Patchett et due to larval attack. The latest research on the sub- al., 2008). Bryant et al., (2010) reported that lolines ject determined that densities above or equal to 125 released by the endophyte Neotyphodium uncinatum adults/m2 are critical to the establishment of pasture present in Festuca pratensis, were effective in con- (Cisternas and Villagra, 2003). Chemical control has trolling Adoryphorus coulonii larvae (Coleoptera: limited effectiveness on adult’s insect, and it is inef- Scarabaeidae) under laboratory conditions. In another fective against the larvae that populate the tillers. Fur- interesting work, Karpyn et al., (2017) compiled in- thermore, organophosphate insecticides present risks formation on the detrimental effect of certain strains on the environmental such as residual effect, exclu- of ryegrass endophytes on the survival and oviposi- sion period and toxicity on pollinators when applied tion of African black beetle. The use of endophytes close to the flowering period (Urrutia, 2005), reason of the genus Neotyphodium that accumulate lolines or for which there is growing interest in the search for another alkaloid in the roots of plants and to reduce new control alternatives (Tampe et al., 2016). In this the growth, development and survival of subterranean sense, the main strategy used to control this weevil would be a potential method for pest control is based on the use of cultivars of Lolium and fes- in grasslands, which would have some advantages cue (Festuca arundinacea) endophyte-infected with such as, effectiveness, low cost and no impact toxic to

Journal of Soil Science and Plant Nutrition, 2017, 17 (1), 91-98 Incidence of Listronotus bonariensis (Coleoptera: Curculionidae) in ryegrass pastures 93

livestock grazing. In Chile, the research on fungal en- 2. Materials and Methods dophytes began in the early 90s. Although, research- ers at the Instituto de Investigaciones Agropecuarias The study was carried out on 12 pastures located be- (INIA) conducted several studies and obtaining valu- tween the regions of Bío-Bío and Los Lagos during able results on the controlling effect of endophyte on the period 2013-2014 and 2014-2015 (Table 1). The L. bonariensis, its current research is outdated. The prospection method used for determining the inci- latest report dates from 2003, and corresponds to the dence of L. bonariensis was a variation of the meth- compilation of the information related to the effect odology proposed by Alzugaray et al., (2010). of the interaction of the Argentine stem weevil and At each selected site, 10 squares (0.3 x 0.3 m) were species of the genus Lolium in milk and meat bovine selected randomly per sampling unit (n=10) with production in southern Chile (Lanuza et al., 2003). 3 replicates each. All plants (100 tillers) were cut Therefore, the aims of this research were to report the at ground level with a scalpel and stored in plas- actual incidence of L. bonariensis and presence of en- tic bags and taken to the Laboratorio de Química dophytes in ryegrass pastures of southern Chile. Ecológica (LQE) of the Universidad de La Frontera.

Table 1. Ryegrass pastures located in different areas of southern Chile during seasons 2013-2014 and 2014-2015. The evaluated samples correspond to the average of the two seasons.

The presence of L. bonariensis was determined as leaves following a modification of the methodology following: a) through immature stages (direct) and proposed by Alarcón et al., (2010). b) by identifying specific damage on the plant (in- The verification of the presence of endophytic fun- direct). The presence and confirmation of immature gi in tissue was checked by the method adapted by stage (eggs and larvae) of L. bonariensis was evalu- Dombrowski et al., (2006). Briefly, 100 tillers ob- ated under a stereoscopic microscope following the tained from different ryegrass pastures were cut and entomological keys by Artigas (1994), while that the the inner epidermis of a leaf sheath was peeled off incidence of the curculionid on the plant was evalu- and placed on a glass slide. Two drops of rose Ben- ated through identification of damage on stems and gal stain were applied to each sample. After 60 s the

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samples will be covered with a cover slip. Finally, The presence of the insect was detected by: a) damage the microscopic examination (40X) was performed, caused to the level of tillers base by the action larvae, identifying as positive those samples presenting typi- b) foliar consumption by adults, and c) presence of cal fungal mycelium. The percentage infection will oviposited eggs into the tissue of the sheath. Although be determinate from rate of samples with endophyte differences in the degree of insect attack and number and the sampled examined. of pastures sampled by region are observed, it shows that this weevil is present in the four southern regions 3. Results and Discussion of the country being regions of La Araucanía and Los Lagos which had a higher presence of the insect. The Figure 1 shows a geographical distribution of L. results for the Región del Bío-Bío showed an lower bonariensis in different ryegrass pastures of south- damage than other regions evaluated (Figure 2). Cato ern Chile zone presented a low level of damage (6.3%) simi- lar to the localities of Chillán and Los Ángeles where there was no presence of individuals or damage by L. bonariensis. The above is because in those zones there are great surfaces of other crops that are hosts of L. bonariensis such as wheat, barley, oats and maize, therefore its presence and incidence in ryegrass is lower than other areas evaluated (Norambuena and Gerding, 1985). The results of the incidence of this insect prospected in the other regions indicated that the most intense attacks by L. bonariensis were found in Region de Los Lagos and in a less extent in regions of La Araucanía and Los Ríos, which is consistent with the report of Cisternas and Villagra (2003). The zones of Región de Los Lagos, Reme- hue, Osorno and Purranque had a similar pasture damage product of action of the L. bonariensis (33, 29 and 30%) (Figure 2). In addition, the average number of immature stages (eggs + larvae) for this region was 268.5/m2, value that exceeds the limits Figure 1. Geographical distribution of damage pro- established by Prestidge et al., (1985) and Cisternas duced by Listronotus bonariensis in different ryegrass and Villagra (2003), who reported 180 and 125 in- pastures in southern Chile. Symbology described in dividuals/m2 respectively, being these values critical the figure indicates different degrees of attack of L. for the survival of a pasture. Similar to the results bonariensis in each zone evaluated during seasons obtained in Región de Los Lagos, in the Región de 2013-2014 and 2014-2015. La Araucanía an average of 105.5 immature stages/ m2 was obtained, which would affect ryegrass pas- tures production. These results do not agree with

Journal of Soil Science and Plant Nutrition, 2017, 17 (1), 91-98 Incidence of Listronotus bonariensis (Coleoptera: Curculionidae) in ryegrass pastures 95

Barrat et al., (2016) who indicated that the average Moreover, it is inferred that the potential for the pro- of L. bonariensis individuals varies between 13-40/m2 duction of forage could be negatively affected be- in New Zealand pastures. These same authors suggest cause L. bonariensis remains present and damaging that climatic conditions and its phenology are the the ryegrass pastures of southern Chile. The micro- main factors that affected fitness and performance of scopic analysis of ryegrass indicated that the high- L. bonariensis. On the contrary in zones of Vilcún est incidence of endophyte fungus was found in the (Región de La Araucanía) and Remehue (Región Maquehue zone (41%, Table 2). In this same area, de Los Lagos) an opposite effect to the rest of the there is a high damage produced by L. bonariensis areas evaluated. In both areas, it was observed that (Figure 2) which would indicate that the no protection the damage produced by L. bonariensis is less than of the endophyte fungus to the plant, possibly due to the amount of collected insects. Possibly this effect is loss of viability of the fungus. The above would be related to the presence of endophytes that act inhibit- explained because as the age of forage seed increases ing the action of the weevil. However, this should be the endophyte fungus loses its viability which possi- evaluated in a future study. Based on the results of bly occurring with the pastures of the Maquehue zone two years of prospecting, it is estimated that in the (Wheatley et al., 2007; Hill and Roach, 2009). The zones of greatest insect attack (Región de La Arau- rest of the areas presented an infection of the endo- canía to Los Lagos), an average of 29.2 individuals/ phyte less than 30% (Table 2). m2 produce about 30% damage.

Figure 2. Average damage and number of immature stages (eggs + larvae) of Listronotus bonariensis in differ- ent ryegrass pastures in southern Chile. Zones evaluated during seasons 2013-2014 and 2014-2015. The zone of Angol (Región de La Araucanía) was only evaluated in the season 2014-2015.

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Table 2. Percentage (%) of infection of endophyte fungus in ryegrass pastures in different areas of southern Chile during seasons 2013-2014 and 2014-2015.

References 4. Conclusions

Alarcón, D., Ortega, F., Perich, F., Pardo, F., Parra, In conclusion, this survey has revealed that the Ar- L., Quiroz, A. 2010. Relationship between radi- gentine stem weevil, L. bonariensis, still affecting the cal infestation of Hylastinus obscurus (Marsh- ryegrass pastures of southern Chile regions. Probably am) and the yield of cultivars and experimental the cause of the damage increase by L. bonariensis lines of red clover (Trifolium pratense L.). Jour- (>30%) is due to a lack controlling effect of endo- nal of Soil Science and Plant Nutrition. 10, 115- phytes fungi, therefore, new control strategies must 125.n be performed. Future studies should addressed to (1) Alzugaray, R., Ribero, A., Silva, H., Stewart, S., prospect the presence of endophytic fungi in the rye- Castiglioni, E., Bartaburu, S., Martínez, J.J. grass pastures evaluated and (2) search for new strains 2010. Prospección de agentes de mortalidad of endophytic fungi that could be more effective in natural de áfidos en leguminosas forrajeras en controlling L. bonariensis. Uruguay. Agrociencia. 14, 27-35.

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