Cognizant of the important role of boron in enhancing carbohydrate transport in plants, this experiment was conducted to determine the interactive effects of foliar boron fertilization and an ectomycorrhizal fungus, Pisolithus tinctorius (Pers.) Coker & Couch on growth and assimilate partitioning in shortleaf pine (Pinus echinata Mill.) seedlings. Our results revealed that the fungal symbiont in concert with boron significantly increased various growth parameters in shortleaf pine at 19 and 25 weeks after emergence. 14C-photosynthates allocated to the needles, stems and roots of 25-week old ectomycorrhizal seedlings increased 50%, 62%, and 116%, respectively over those of noninoculated seedlings. Foliar boron, however, did not enhance 14C partitioning to the roots of ectomycorrhizal or nonmycorrhizal seedlings.

The application of certain micronutrients as an effective approach to enhance mycorrhizal colonization in some plant species is well documented (Dixon et al., 1981). A similar study (Atalay et al., 1988) showed that fertilization with boron increased the total carbohydrate content of mycorrhizal roots of 16 week-old shortleaf pine seedlings. Root sugar contents and ectomycorrhizal development were found to be correlated in loblolly pine (Pinus taeda L.) seedlings (Marx et al., 1975). Boron nutrition has also been documented to affect the translocation of sugars to the roots (Van de Vender and Currier, 1977). Although previous reports have described the distribution of 14C in roots of various mycorrhizal plant species (Bethlenfalvay et al., 1982; Jones et al., 1991; Klironomos and Kendrick, 1993; Koch and Johnson, 1984; Pang and Paul, 1980; Snellgrove et al., 1982; Soderstrom et al., 1988), a study dealing with the influence of boron on the allocation of 14C-photoassimilates to the root systems of ectomycorrhizal shortleaf pine would expand our understanding of how boron affects ectomycorrhizal development in pine.

The present study examined the effects of foliar boron fertilization and an ectomycorrhizal fungus [Pisolithus tinctorius (Pers.) Coker & Couch] inoculation on growth and 14C-photosynthate partitioning to various tissues of shortleaf pine seedlings, with emphasis on 14C distribution to the root systems. Specifically, the hypotheses tested were: (a) 14C allocation to the root systems will increase with inoculation and, (b) 14C fixation will be enhanced by foliar boron application.

Plant Culture--Vegetative inoculum of P. tinctorius was cultured in an autoclaved mixture of peat and vermiculite (1:10, v/v) according to previously described procedures (Marx and Bryan, 1975). After 25 weeks of growth, the inoculum was leached repeatedly with deionized water to remove unused nutrients, then used immediately.

Full-sib shortleaf pine seeds were surface-sterilized for 5 min in 30% H2O2, then rinsed three times with sterile, deionized water. Seeds were further soaked in sterile, deionized water for 48 hours at room temperature, drained of excess water, and stratified at 4C for two months.

The growth medium of noninoculated seedlings consisted of a mixture of sandy loam soil, peat, perlite, and vermiculite (1:1:1:1, v/v/v/v) sterilized with Dowfume MC-2 (98% methyl bromide, 2% chloropicrin; Dow Chemical Co., Midland, MI, USA) at a rate of 680 g m-3. Inoculated seedlings were grown in a 1:8 (v/v) mixture consisting of washed vegetative inoculum of P. tinctorius and sterile growth medium.

Seeds were sown in four-cavity Spencer-LeMaire book planters (500 cm3), which had been sterilized with 10% sodium hypochlorite solution. Three seeds were planted per cavity. Each cavity contained 500 cm3 of growth medium. After emergence, seedlings were thinned to one per cavity.

Plants were maintained in a greenhouse with the photoperiod extended to 16 hours with a high pressure sodium lamp (1000 W G.E. Lucalox). Using a LI-COR 6200 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA), lamp height was adjusted periodically to provide a photon flux density of 700 mol m-2 s-1 at the top of the seedling canopy. Ambient greenhouse temperature conditions ranged between 25-32C during the day and 20-24C at night. Each plant received 20 ml of full strength, modified Hoagland's solution (Bonner and Galston, 1952) once a week starting at 2 weeks after emergence. Plants received deionized water as needed.

Growth Analyses and Mycorrhizal Assessment--Nondestructive determinations of seedling height, number of branches, and stem diameter (at the root collar) were made at 19 weeks after emergence. Prior to 14CO2 exposure at 25 weeks after emergence, 4 randomly selected seedlings per treatment combination were set aside for mycorrhizal assessment. Roots were cleared and stained with 0.1% Ponceau S (acid red 112) in 10% acetic acid following the procedures of Daughtridge et al. (1986b). Mycorrhizal infection on cleared, stained roots was determined by counting the number of primary laterals, the number of primary laterals with mycorrhizal structures, and the number of infection points (ectomycorrhizal short roots) per infected lateral. Growth parameters at 25 weeks after emergence were obtained from seedlings prior to 14CO2 exposure.

Experimental Design--Experimental units were arranged in a 2 (inoculation treatments) x 2 (boron concentrations) factorial arrangement in a completely randomized design (CRD) with 4 replications. Each book planter, consisting of 4 seedlings constituted a treatment replicate. Beginning at 5 weeks and continuing until 8 weeks after emergence, a boron solution (25 g B ml-1) containing 0.1% (w/v) Tween 20 was applied once a week as a foliar mist using a hand sprayer. From 9 weeks until 20 weeks after emergence foliar boron mist was applied twice a week. Approximately 15 ml of spray solution were needed to wet each seedling adequately. Aluminum foil was used to cover around the stem base to prevent spray mist from reaching the growth medium.

Labeling with 14CO2--14C labeling began at 25 weeks after planting. The plexiglass chamber (0.27 m W x 0.28 m H x 0.53 m L) used for labeling was made of two components: a base with 32 (0.019 m diameter) holes through which pine shoots were inserted and sealed with a putty-like compound (Apiezon Q, American Scientific Products, McGaw Park, IL, USA) around the stem to prevent CO2 from escaping and a double-walled cover which enclosed the shoots. Water was circulated between the two walls of the chamber to prevent heat build-up during labeling. Also ports at the top of the chamber facilitated the generation of 14CO2 in the chamber and allowed electrical access to a small fan for mixing air in the chamber. The 14CO2 was generated by dropping HCl into a vial in the chamber containing NaH14CO3 (specific activity, 56.5 mCi/mmol, ICN Radiochemicals, Irvine, CA, USA). Seedlings were not removed from the book planters during labeling. Thirty-two seedlings were labeled at a rate of 10 Ci per seedling with the chamber left in place for 3 hours.

Harvesting Procedures--Immediately after labeling some shoots were severed at the stem base and frozen in liquid nitrogen. Likewise roots were washed carefully in water and dipped in liquid nitrogen. Plant parts were separated into needles, stems, and roots; lyophilized for at least 48 hours; and then weighed. Subsequent samplings were made at 24 hours after labeling.

Lyophilized samples were ground in a Wiley mill equipped with a 180 m sieve. One hundred mg of each ground plant tissue sample were combusted in a Tri-carb Sample Analyzer (Model 306, Packard Instrument Co.) and then mixed with 10 ml of Insta-Gel scintillation cocktail. 14C content of samples were determined using a liquid scintillation counter (Beckman Model 1701S).

Ectomycorrhizal colonization resulted in significant increases in stem diameter, height, and number of branches of shortleaf pine seedlings at 19 weeks after emergence (Table 1). Also, boron fertilization increased the mean stem diameter and height, but not number of branches, of both mycorrhizal and nonmycorrhizal seedlings. After 25 weeks of growth, inoculated seedlings had more branches and greater stem diameter (Table 2). Branch, needle, stem, and root dry biomass were also increased following inoculation. Moreover, foliarly applied boron was associated with an increase in the number of branches, and enhanced branch, needle, and stem dry weights, but had no effect on stem diameter and root dry weights.

Percentage of infected roots increased by more than twofold in inoculated seedlings, compared to noninoculated controls. Boron fertilization, however, did not improve mycorrhizal colonization (Table 2).

At 24 hours after labeling, 14C photosynthates allocated to the needles, stems, and roots of inoculated seedlings increased by 50%, 62%, and 116%, respectively, over those of noninoculated seedlings (Table 3). Foliar boron fertilization had no significant influence on 14C partitioning, although the magnitudes of the allocated 14C were consistently higher in boron-fertilized seedlings than in unfertilized controls.

Table 1. Stem diameter, height, and branch numbers of 19-week old noninoculated (NM) and inoculated (M) shortleaf pine seedlings foliar-fertilized with 0 (-B) or 25 g ml-1 (+B) boron.

Means in a row followed by a common letter are not significantly different from each other according to LSD test (P=0.05). Each value represents the average of 16 replications.

Table 2. Mean growth responses and mycorrhizal colonization of 25-week old noninoculated (NM) and inoculated (M) shortleaf pine seedlings foliar-fertilized with 0 (-B) or 25 g ml-1 (+B) boron.

Means in a row followed by a common letter are not significantly different from each other according to LSD test (P=0.05). Each value represents the average of 4 replications.

Table 3. Total 14C radioactivity (cpm) at 24 hours after labeling in various tissues or 25-week old noninoculated (NM) and inoculated (M) shortleaf pine seedlings foliar-fertilized with 0 (-B) or 25 g ml-1 (+B) boron.

Means in a row followed by a common letter are not significantly different from each other according to LSD test (P=0.05).

Inoculation with the ectomycorrhizal symbiont, P. tinctorius, significantly increased the various growth parameters in shortleaf pine seedlings both at 19 and 25 weeks after emergence. Foliar fertilization with 25 g ml-1 boron enhanced most of the growth responses in both mycorrhizal and nonmycorrhizal seedlings. These observations suggested that foliar boron affected both the fungal symbiont and/or plant host-symbiont combination as previously reported (Daughtridge et al., 1986a; Mitchell et al., 1987). Particularly striking in this study was the marked increase in branch number and biomass of boron-fertilized, inoculated seedlings, an observation that has never been reported for this species. A previous report (Schon and Blevins, 1987) of a boron-mediated increase in the number of soybean branches and pods on lateral branches is compatible with our observation. In fact, studies (Allen et al., 1980; Dixon et al., 1988) have attributed the increased biomass in seedlings infected with vesicular-arbuscular mycorrhizae (VAM) to an elevated cytokinin flux from the roots to the shoots. In view of the generally accepted role of cytokinins in enhancing cell division, we suggest that the increased branch number and biomass of mycorrhizal shortleaf pine could be attributed to a fungal symbiont-mediated increase in root cytokinin production. We hope to explore this line of research in the very near future.

Since mycorrhizal fungi depend on the host plant for carbohydrates, benefits could be derived from any increased photosynthate flux to the roots. Numerous studies (Cairney et al., 1989; Koch and Johnson, 1984; Snellgrove et al., 1982; Wang et al., 1989) have shown significantly more fixed carbon translocated from shoot to root in inoculated plants than in noninoculated plants. It was evident in this study that the significant increase in 14C photosynthates partitioned into the tissues of inoculated seedlings, especially into the ectomycorrhizal roots, resulted in more than a twofold improvement in mycorrhizal colonization. Contrary to our hypothesis, foliar boron did not enhance 14C allocation to the root systems. This finding is consistent with a previous observation indicating that foliar fertilization with boron did not increase carbohydrate content of mycorrhizal roots in shortleaf pine seedlings (Atalay et al., 1988). More 14C accumulation had been previously demonstrated to occur in young, rather than in older, mycorrhizas (Cairney et al., 1989). Perhaps the effect of boron on assimilate transport to the roots could have been more pronounced if 14C labeling coincided with the time when majority of the mycorrhizal roots were acting most strongly as carbon sinks. More detailed experiments are being planned to determine 14C allocation to the roots at various periods after fungal symbiont inoculation.

We gratefully acknowledge the financial support of the Department of Biology, Jackson State University Howard Hughes Medical Institute Undergraduate Sciences Initiative Program. We also thank Dr. John D. Hesketh, Photosynthesis Research Unit, USDA-ARS, Urbana, IL 61801, for his critical review of the manuscript.

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