The Association of a Longidorus Species with Stunting and Root Damage of Loblolly Pine (Pinus taeda L.) Seedlings

Fraedrich, S. W., and Cram, M. M. 2002. The association of a Lungidorus species with stunting and root damage of loblolly pine (Pinus ruedu L.) seedlings. Plant Dis. 86803-807. A Longidorus species was consistently associated with patches of stunted and chlorotic loblolly pine seedlings at a forest-tree nursery in Georgia. Seedlings from affected areas had poorly developed root systems that lacked lateral and feeder roots. Longidorus population densities in composite soil samples from the margins of patches ranged from 9 to 67 nematodes per 100 cm3 of soil. In a growth chamber experiment, seedling root dry weight decreased with respect to the initial Lungidorus dose as well as the final Lungidorus populations in containers. The dry root weight of seedlings were 0.117, 0.090, 0.066, and 0.065 g in containers initially infested with 0, 50, 100, and 200 Longidorus, respectively. Lateral and fine roots were lacking on seedlings at the highest doses. Populations of Longidorus increased in all containers during the experiment. Damage to loblolly pine seedlings caused by Longidorus is a previously undescribed problem in southern pine nurseries. Proper diagnosis of the problem by nematode testing laboratories may require the use of extraction techniques specific for larger nematodes such as Longidorus. Many species of plant parasitic nematodes are associated with roots of conifers (lo), and some can cause significant damage (9,11,12,16). Nursery seedlings are particularly vulnerable to damage by nematodes because of the continuous culture of single species, use of irrigation to maintain soil moisture, and maintenance of high soil fertility for optimal plant growth (12). Fumigation has been used routinely for more than 40 years in many southern forest-tree nurseries to control weeds, insects, fungal pathogens, and nematodes. However, the most widely used fumigant, methyl bromide (6), is scheduled to be phased out by 2005. Many aspects of nursery operations have changed since the 195Os, and there is presently a lack of information about soilbome diseases that may affect pine production in southern nurseries. Areas of stunted pine seedlings have been periodically observed in fields at the Flint River Nursery (Byromville, GA) since its establishment in 1987. During 1998 and 1999, patches of stunted and chlorotic loblolly pine (Pinus rue& L.) seedlings occurred in one field at this nursery. Soil samples from the affected areas were forwarded to a nematode testing laboratory for evaluation of plant parasitic Corresponding author: Stephen Fraedrich E-mail: [email protected] Accepted for publication 4 April 2002. Publication no. D-2002-0521-02R This article is in the public domain and not copy rightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2002. nematodes, and seedlings were sampled for fungal pathogens, but a definitive cause of the problem could not be determined. Sections of the field affected by the disease were fumigated in the spring of 2000, and the disease was not observed in the pine seedling crop produced in these sections during 2000. In July 2000, we again observed patches of stunted loblolly pine seedlings but in different sections of the field. These sections contained a fumigation study (3) that was in its third year of continuous pine seedling production. Seedling damage was observed in nonfumigated control plots, and soil in these plots had not been fumigated since October 1993. Many seedlings were chlorotic and severely stunted in affected areas (Fig. 1A). Compared to healthy seedlings, stunted seedlings had poorly developed root systems that lacked lateral, feeder, and mycorrhizal roots (Figs. 1B and IC). Soil samples were forwarded to a nematode-testing laboratory where lesion (Prufylenchus sp.) and ring (Criconemella sp.) nematodes were found at low levels in areas with diseased seedlings as well as areas with healthy seedlings. Roots were plated on various agar media, and Fusarium spp. were routinely isolated from both healthy and diseased seedlings. A Rhizoctonia-like fungus was also occasionally isolated from roots of stunted seedlings in some patches. Upon closer inspection of the unwashed roots in water under a dissecting microscope, we routinely observed needle nematodes (Longidorus sp.) associated with diseased seedlings. This nematode had not been reported by the nematode testing laboratory. We summarize in this paper our findings of a survey for a Longidorus sp. in affected areas of seedbeds at the Flint River Nursery and results of a growth chamber experiment to determine the effect of Longidorus sp. on loblolly pine seedlings. MATERIALS AND METHODS Field survey. Seedlings and soil were sampled in four areas that contained patches of stunted loblolly pine seedlings. Each patch occurred in a separate bed within a 12-seedbed area of one field. Patches ranged from 3 to 9 m long and were one seedbed (1.2 m) wide. Samples of seedlings with soil attached were collected from each of the four patches in August and October 2000. In August, diseased seedlings were collected at patch centers and margins, and healthy-appearing seedlings were collected at distances of 1.5 and 3.0 m outside the margins of patches (seven sample locations per patch). In October, seedlings were collected at patch margins and at distances of 1.5 m outside the patch margins (four sample locations per patch). At each sample location and sample time, 10 to 25 seedlings were lifted. Seedlings with attached soil were placed in plastic bags. A 25 g sample of soil from the root zone of seedlings at each sample location was used for nematode extractions. At patch centers, only 8 to I5 g of soil was used because of the sparseness of seedling roots and lack of associated soil. The population density of Longidorus in these samples was expressed on a 25 g weight basis. Nematodes were extracted using the technique of Flegg (5) but the technique was modified such that 90 pm aperture sieves were used in place of 150 pm sieves. In addition, nematodes and debris from sieves were placed in water under a dissecting scope, and nematodes were counted directly without the use of the Baermann funnel apparatus. Composite soil samples were collected with a soil sampler in September and again in October 2000 from the centers, margins, and 1.5 m outside the margins of each of the four patches (five sample locations per patch). Soil samples from each location consisted of 5 to 6 soil cores (2.5 cm diameter to a 1.5 cm depth). Nematodes were extracted from 200 cm3 of soil using the technique of Flegg (5) with 90 pm aperture sieves as previously noted. In addition, Kimwipes (Kimberly-Clark Corp., Roswell, GA) were used instead of 90 pm aperture nylon screen in the Baermann funnel apparatus. After 48 h, about 2.5 ml of water with nematodes was removed from the Baermann funnels, and LongiPlant Disease /July 2002 8 0 3 dorm were counted under a dissecting microscope. Debris with nematodes was washed from the Kimwipes into a petri dish, and remaining Longidorus were counted under a dissecting microscope by systematically sorting through the debris. Growth chamber experiment. The objective of the experiment was to examine Fig. 1. A, patch of stunted loblolly pine seedlings at Georgia nursery. Root systems of healthy B, and Longidorus survey. In August 2000, diseased C, IO-week old seedlings. Longidorus population densities were 8 0 4 Plant Disease / Vol. 86 No. 7 the effect of the Lmgidorus sp. on growth of loblolly pine seedlings. Longidorus were extracted from soil using the technique of Flegg (5) with the 90 pm aperture sieves and without additional modifications. The nematodes were hand picked, placed in water in groups of 50, and stored at 5°C for up to 48 h before introduction into containers. Soil was obtained from areas of the nursery not affected by disease and was microwaved in 2 kg batches for 8 min. Approximately 330 g of soil was placed in each container (7 cm high by 10 cm wide). Loblolly pine seeds were surfaced sterilized with hydrogen peroxide (30%) for 60 min (l), rinsed with sterile distilled water, and stratified for 30 to 60 days. Seeds were germinated under sterile conditions, and five germinated seeds were transplanted to containers with microwaved soil. Needle nematodes were added to containers at rates of 0, 50, 100, or 200 individuals per container, and there were four replications of each nematode dose. The seedlings were placed in a growth chamber at 22°C with a 14-h photoperiod and were watered every 1 to 3 days as needed. At the end of 22 weeks, seedlings were removed from containers and dried at 80°C for 48 h. The dry root and shoot weights of each seedling was determined. The final population of Longidurus in containers was determined using the extraction technique of Flegg (5) with the 90 pm aperture sieve and without additional modification. The experiment was established as a completely randomized design with four treatments (Longidorus doses) and four replications (containers) per treatment. The effect of the initial Lmtgidorus dose and final Longidorus populations on seedling root and shoot dry weights was determined by regression analysis (4). A nonlinear, negative exponential model was used to characterize the relationship between the initial Longidorus dose and seedling root dry weight. Parameter estimates were determined using PROC NLIN (The SAS System for Windows, Version 8.01, SAS Institute, Inc., Cary, NC). The criteria for fit of the model were based on the mean square error (MSE), the significance of the overall regression, and a lack of fit analysis. Linear regression using PROC REG (The SAS System for Windows, Version 8.01) was used to analyze the relationship between initial Longidorus dose and seedling shoot dry weight, and relationships between final population of Longidorus per container and seedling root and shoot dry weights. Longidorus populations in containers at the end of the experiment were analyzed among treatments using an analysis of variance and Tukey’s honest significant difference (HSD) procedure (0~ = 0.05) for mean separation (8).