National Canola Research Program

National Canola Research Program

Managing blackleg: do flea beetles vector blackleg in canola?

PI: Luis del Rio, Department of Plant Pathology, NDSU Fargo, ND 58108

Co-PI:Janet Knodel, Department of Entomology, NDSU Fargo, ND 58108

Co-PI:Scott Halley, NDSU-REC Langdon. Langdon, ND 58249

Introduction

Until recently, blackleg of canola, caused by the fungus Leptosphaeria maculans (Desm.) Ces. & de Not., was considered to be under control in North Dakota thanks to the extensive use of resistant cultivars. The discovery of new L. maculans strains in recent years (Bradley et al., 2005; Chen and Fernando, 2006) was an indication that blackleg may be coming back as a serious threat to the canola industry of the region. A survey of L. maculans phenotypes prevalent in North Dakota in 2007 and 2008 indicated that the population was shifting from a pathogenicity group (PG) 2 dominated to a more diverse population (Mazurek, 2011). Strains of newly detected PG-3 and PG-4 are capable of attacking most commercial canola cultivars in use (Marino and del Rio Mendoza, 2010). This is an indication that blackleg is no longer a disease under control. Blackleg outbreaks caused by PG-4 strains that were detected in 2009 (del Rio et al., 2012) confirm this observation.

L. maculans infects plants at any stage of its development; however, yield robbing cankers are usually formed at the base of the stems only when infection takes place before the plants pass the third-to-fifth leaf stage (Marcroft et al., 2005). The initial symptoms of infection are expressed as necrosis around the area where the pathogen penetrated; however, once the pathogen reaches the vascular tissues and starts moving into the stem external symptoms are no longer visible. Later on, however, infected plants may exhibit reduced growth and eventually cankers are formed at the base of the stem. The pathogen survives on infected residues on the field and from there it gets transferred into seedlings by wind, rain splash and perhaps insect feeding activity.

The crucifer flea beetle (Phyllotetra cruciferae) is one of the most important insect pests affecting establishment and yield of canola. This small insect survives the winter as an adult in leaf litter of shelterbelts or in grassy areas and in rare occasions on canola stubble (Knodel and Olson, 2002). When canola seedlings emerge, flea beetles feed on seedlings causing small wounds on the cotyledons. Severe insect pressure could kill seedlings and results in significant yield reductions (Lamb, 1984). Flea beetles are strong fliers; however, in rainy conditions they usually walk or hop resulting in aggregated distributions in the fields (Knodel and Olson, 2002). Flea beetles that survived the winter lay eggs in the soil near canola roots sometime in June (Burgess, 1977). The new generations of flea beetles become adults in late summer and are the ones that will feed on the canola seedlings the next year (Westdal and Romanow, 1972). Wounds caused by the feeding habits of the crucifer flea beetle on canola seedlings could facilitate the entrance of organisms like L. maculans into plant tissues. In the absence of wounds, L. maculans spores penetrate into plant tissues mainly through stomata (Hua et al., 2005). In spite of the potential association between these two pests, L. maculans and P. cruciferae, very little is known about their potential interaction. However, it would be logical to conclude that at the very least flea beetle damage may increase the risk of infection by blackleg spores. We propose to study this association as a means to identify better ways to manage both pests.

Current work

Penetration of blackleg into plant tissues:

Preliminary experiments to study the association between flea beetles and blackleg were conducted in field and greenhouse conditions in the summer and fall of 2011 with support from the Northern Canola Growers Association. A replicated trial conducted at the NDSU REC Langdon used mesh screens to keep pairs of experimental plots with specific levels of flea beetle infestation until canola plants were at the sixth-leaf stage. One plot of each pair was inoculated with L. maculans spores by spraying a 103 spore suspension on plants at the third-leaf stage, the other was not. Blackleg incidence was measured prior to swathing. Results suggested that in the absence of flea beetles plots that were inoculated with spores of L. maculans had significantly higher incidence of blackleg than plots that were not inoculated. No significant differences were observed between inoculated and non-inoculated plots that had different densities of flea beetles in them (Figure 1).

Figure 1. Blackleg incidence (%) in canola plots infested with several flea beetle densities and inoculated or not with spores of L. maculans at the third-leaf stage.

Flea beetles that were captured from that field in Langdon were taken to a greenhouse facility in Fargo. Canola seedlings that had been previously exposed to flea beetles and had feeding wounds were sprayed with a 107 spore suspension of L. maculans. Seedlings that had not been exposed to beetles were also sprayed. These seedlings and other that were inoculated by mechanically wounding cotyledons were incubated overnight in moist chamber and transferred to greenhouse room. At flowering time plants were examined for blackleg damage. Results of this trials indicated that seedlings exposed to flea beetles and L. maculans spores had significantly higher (P=0.05) amounts of blackleg than those exposed to spores only (Figure 2). Incidences of blackleg for the flea beetles + spores treatment also were similar to the spores + wounded cotyledons (Figure 2).

Figure 2. Effect of the combined activity of flea beetles and inoculation of L. maculans spores on blackleg incidence at cotyledon stage in greenhouse conditions.

While the preliminary data presented here strongly suggest that the crucifer flea beetle could enhance the damage caused by blackleg, almost nothing is known about the way these two organisms interact. Questions like, are L. maculans spores transported by the flea beetles, where do they carry them and how do these two species interact, or even is P. cruciferae the only species that interacts with L. maculans?, need to be answered. Interactions between insects and plant pathogens have been known to exist for long time. Perhaps one of the most commonly known are related to the way aphids and other plant sap suckers vector viruses; however, insects can also vector fungi and bacteria. For example, elm bark beetles (Scolytus spp.) transport spores of Ceratocystis ulmi, causal agent of Dutch elm disease, on their bodies (Gibbs, 1978) whereas the corn flea beetle, Chaetocnema pulicaria transports the bacterium that causes Stewart’s wilt of corn in its guts (Esker and Nutter, 2003). This proposal outlines research intended to validate the results obtained in the preliminary trials presented here, and to determine their relationship.

Objective

The objective of this project is to determine whether crucifer flea beetles affect the development of blackleg epidemics in North Dakota.

Rationale and significance

Blackleg epidemics have been increasing steadily during the past few years in North Dakota in part caused by an increase in genetic diversity of the L. maculans populations and lack of genetic resistance of commonly planted canola cultivars to new strains of blackleg. Preliminary findings strongly suggest that the feeding habits of the crucifer flea beetles can enhance the risk of blackleg occurrence by providing more injury points of entry for the fungus to infect plants. The enhanced risk of blackleg infection means that fewer spores may be needed to infect seedlings. This situation may have not been noticed before because most cultivars were resistant to blackleg strains that are no longer the most predominant. Findings of this research could have implications on flea beetles and blackleg management in canola.

Approach

Field studies:

Replicated experiments will be installed in Langdon to estimate the amount of blackleg that develops on caged plots that contain different densities of flea beetles. Flea beetles will be collected in the spring using pheromone-baited traps (Soroka et al., 2005) and sweep net samples from an early planted canola plot nearby. Flea beetles will be aspirated and counted to the desired densities (1, 10, 100, and 500 per cage), placed in vials and then quickly released into experimental caged plots. Samples of flea beetles will be preserved in ethanol for identification and confirmation of species during collections.

Treatments include L. maculans inoculated and non-inoculated canola with four different flea beetle densities (0, 10, 100, 500). In this study, all plots will be covered with cages immediately after planting. One week after plant emergence, pairs of plots in each replication will be infested with similar number of flea beetles. Each plot will be assessed for flea beetle damage by collecting data once a week for a three week period beginning at the 1-leaf stage (first true leaf expanded). Variables measured for each plot will be plant stand, percent plants damaged, flea beetle injury, seedling vigor, and percent canopy cover. Plant stand will be measured by counting the number of plants in six feet of row (three feet in two adjacent center rows). Plant stand will be converted to plants/ft2. Flea beetle injury will be measured using a 1-6 scale: 1 = 0-3 pits per seedling, 2 = 4-9 pits per seedling, 3 = 10-15 pits per seedling, 4 = 16-25 pits per seedling, 5 = >25 pits per seedling and 6 = dead seedling. Seedling vigor will be estimated using a 1-10 scale, with 1 being lowest vigor and 10 being highest vigor. Percent canopy cover will be visually estimated as the percent canopy closure over the entire plot. Seedling vigor and percent canopy cover will be measured only on the third collection date.

Two weeks after planting one of the plots at each level of beetle infestation will be sprayed with L. maculans spores at a concentration of 105 per ml without opening the cages. This application will be repeated two more times at five day intervals. The cages will be kept on the plots until plants have reached the sixth-leaf stage. At that time herbicides will be applied to control weeds. Before swathing blackleg incidence and severity will be assessed on 40 plants per plot. Yield will be calculated after harvest.

Greenhouse studies:

The interactions between flea beetles and blackleg will be studied in controlled conditions. Replicated experiments will be conducted to establish the association between the degree of damage caused by flea beetles on canola cotyledons and the risk of blackleg development in the presence of different L. maculans inoculum concentrations. Similarly, replicated experiments will be conducted to determine whether the flea beetle can vector the pathogen from one plant to another. To accomplish this, field-captured insects will be forced to feed on canola seedlings infected with blackleg or not and then moved into non-exposed seedlings. Disease incidence will be measured in the second group of plants. To determine whether the insect harbors L. maculans over the winter, samples from the first waves of flea beetles coming from overwintering refuges will be collected and force to feed on greenhouse-grown canola seedlings. Disease development will be estimated at flowering time. Also, a grow-out test will be conducted using insects collected in this first wave. The test will be conducted by placing insects on V8 medium amended with antibiotics to determine whether L. maculans grows on medium.

Expected results

The studies proposed in this document will help us determine whether flea beetles can increase the risk of blackleg development. We should be able to determine whether flea beetles can vector the disease from one plant to the next and estimate the risk of that to occur. We will be able to quantify the risk of blackleg development using L. maculans inoculum concentration and flea beetle activity as variables.

Pitfalls and limitations

Field trials have higher risk than trials conducted under controlled conditions of being botched up by bad weather conditions, which in the case of this study could be expressed as delays in planting and lower survival rates of flea beetles among others. We are prepared to conduct this study for at least two years. A third year could be sought if bad weather do prevent us from conducting the study as planned. Increasing the number of locations where the experiment will be installed will represent a significant increment in the cost of conducting the experiment and will not reduce the negative impact weather may have on disease development. Greenhouse trials are less affected by weather; however, they will depend on availability of flea beetles.

Project timeline

This project will have duration of two years. The field trials will be conducted at the NDSU Langdon REC. Planting will be conducted in early May of each year (2012 and 2013). Flea beetles will be collected starting approximately one week prior to planting to secure obtaining beetles that have not fed on canola seedlings. Plant infestation with flea beetles and blackleg inoculations will be conducted within the first 35 days of the growing season. Good commercial agronomic practices will be followed to secure growth of the plants. Disease plants will be evaluated when they approach physiological maturity. Greenhouse activities will start two weeks prior to the time when the first collection of flea beetles is scheduled. Repeated canola plantings will be made to secure a fresh supply of canola seedlings. Disease reaction and evaluation will be conducted in greenhouse when plants reach late flowering stages in each year.

Outreach/ Extension activities:

Findings of this project will be shared with canola growers during field days and at commodity-sponsored meetings during the fall and in the winter months. Also, results of this research will be presented as scientific meeting also be shared with the scientific community through presentations at professional meetings and industry/professional publications.