Plant and Soil 233: 85–94, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
85
Inoculation with arbuscular mycorrhizal fungi enhances growth of Litchi chinensis Sonn. trees after propagation by air-layering David P. Janos1,3 , Michelle S. Schroeder1 , Bruce Schaffer2 & Jonathan H. Crane2 1 Department
of Biology, University of Miami, P.O. Box 249118, Coral Gables, FL 33124-0421, USA Research and Education Center, University of Florida, 18905 S.W. 280 Street, Homestead, FL 330313314, USA 3 Corresponding author∗ 2 Tropical
Received on 23 August 2000. Accepted in revised form 13 February 2001
Key words: Glomales, mineral nutrition, phosphorus fertilization, Sapindaceae, soil-free medium, stem cuttings.
Abstract Lychee (Litchi chinensis Sonn.) is typically propagated by air-layering mature tree branches which are potted in fertilized, soil-free media after cutting. The size of these branches, low phosphorus retention by pot substrates, and fertilization all might combine to preclude benefits of arbuscular mycorrhizas to lychee. In order to examine the potential of lychee to benefit from arbuscular mycorrhizas in an agriculturally realistic context, lychee air-layers were grown for 469 days in ca. 95-l pots of soil-free substrate inoculated with field-collected arbuscular mycorrhizal roots or not at two different levels of phosphorus fertilization. High phosphorus fertilization (a one-time addition of ca. 1.32 g l−1 slow-release triple-superphosphate) had no detectable effects on mycorrhiza formation, lychee survival, net CO2 assimilation, or growth. Inoculation with indigenous South Florida arbuscular mycorrhizal fungi improved leaflet expansion as early as 120 days after inoculation, and subsequently enhanced height growth and leaf production but did not affect stem diameter growth, net CO2 assimilation, or survival. At harvest, although mycorrhizal colonization was low (average 7.4% colonized root length), mycorrhizal plants had 39% higher aboveground dry weight than control plants. Below-ground dry weights did not differ, but inoculated plants had lower fine root to leaf dry weight ratios than control plants. Leaflets of inoculated plants had higher concentrations of P, K, Cu, and Zn, and lower concentrations of Ca, Mg, and Mn than those of control plants, but total Kjeldahl nitrogen and iron concentrations did not differ significantly 10 months after inoculation. Mycorrhiza enhancement of lychee growth occurred even though phosphorus clearly was not limiting for growth. Our observations suggest that in this soil-free medium, arbuscular mycorrhizal fungus enhancement of copper and iron nutrition improved lychee growth.
Introduction It is well established that arbuscular mycorrhizas (AM, used generically to encompass both vesicular– arbuscular and strictly arbuscular mycorrhizas) can enhance the growth of seedlings of many tropical tree species (e.g., Janos 1980), including species of commercial fruit trees (Read 1993), in low phosphorus soil. It is less certain, however, that AM will enhance the growth of trees propagated from large stem ∗ Tel.:
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cuttings or air-layers (girdled, rooted branches) that involve considerable woody tissue containing abundant carbohydrate reserves (Menzel et al., 1995) and possibly substantial mineral nutrients. Cooperband et al. (1994) found that AM inoculation of large cuttings (ca. 0.4 m tall) of Erythrina berteroana Urban, a tropical legume tree, had a negative effect on shoot growth 6 months after planting. They attributed this to adequate mineral nutrient reserves in the woody tissue such that mycorrhizas imposed a carbon cost without a recompensing benefit to the plant. Habte and Byappanahalli (1994) observed that 0.18×18 mm diameter cuttings of cassava, which on average contained
86 20.2 mg phosphorus, responded very little to mycorrhizal inoculation, but that 21×9.2 mm diameter cuttings containing 2.0 mg P responded well. Moreover, AM may not enhance plant growth in soil-free potting mixes if there is low retention of phosphorus by the substrate such that extraradical hyphae of the fungi provide little advantage for acquisition of phosphorus beyond that provided by fine roots and root hairs alone (Biermann and Linderman 1983). Lychee (Litchi chinensis Sonn.), a sapindaceous, commercial tropical fruit tree, forms arbuscular mycorrhizas (Pandey and Misra, 1971; Koske et al., 1989) and root hairs. It sometimes produces ‘tuberculate’ ultimate rootlets that are similar to those of Podocarpaceae (e.g., Baylis et al., 1963). Koske et al. (1989) noted that tuberculate rootlets appear to have been confused with glomalean fungus sporocarps. Coville (1921) speculated that AM might be indispensable to lychee, but available data concerning growth effects are equivocal (Menzel and Simpson, 1987). Pandey and Misra (1975) examined the effect of AM inoculation on lychee in pots of sterilized soil, but all control plants without mycorrhizas died. Although this suggested strong dependence by lychee upon AM for survival, it precluded determination of effects on growth. Lychee recently has become an economically important crop in South Florida (Degner et al., 1997). Because it does not come true from seed, grafting and budding are not reliable, and because a relatively high proportion of seedlings may fail to grow (Pandey and Misra, 1975), propagation is typically by air-layering. After air-layers (also called ‘marcots’) are cut from source plants, they generally are potted in soil-free media for several months to allow fine roots to develop from coarse adventitious roots. Soil-free potting media may not expose these fine roots to arbuscular mycorrhizal fungi (Graham and Timmer, 1984). Moreover, air-layers frequently are fertilized with phosphorus (J. Crane, University of Florida, pers. commun.) which might suppress mycorrhiza formation (Graham and Timmer, 1984, 1985; Same et al., 1983). We conducted an experiment to examine the effects of AM on the growth of air-layered lychee trees in a soil-free potting medium with and without high phosphorus fertilization. The objectives were to determine if: (1) field-collected chopped root inoculum that represents a mixture of glomalean fungus species indigenous to South Florida could enhance lychee tree growth following air-layering and transplant into
a soil-free medium, and (2) high phosphorus fertilization would alter plant response to AM.
Materials and methods Lychee trees (cv. Mauritius) were propagated by airlayering branches of mature trees in an orchard (Star Groves, Homestead, FL). Air-layers were started in mid-September, 1997. Air-layered branches with roots were cut from the trees on 2 February 1998, and were pruned to reduce shoot height and leaf area (at the initial measurement on 9 February 1998, the air-layers averaged 0.394 m in height, 11 mm in diameter, and had 6.5 leaves). They were planted in a fumigated potting medium in surface-sterilized 94.5 l black polyethylene pots. The pots were disinfested by washing them with bleach (5% NaOCl). The potting medium consisted of: 4 parts Florida sedge peat, 4 parts pine bark, 2 parts saw dust, and 1 part coarse sand (by volume) with a pH (1:2 paste) of 5.9. The potting medium was fumigated beneath a polyethylene tarp with a mixture of methyl bromide (67%) and chloropicrin (33%) at a concentration of 2.73 kg m−3 for 5 days prior to being aired for approximately seven days before dispensing to pots. Treatments were a factorial combination of mycorrhizal inoculation (+M) or none (−M), and low phosphorus fertilization (LP) or high phosphorous fertilization (HP). The experiment design was a randomized complete block with 10 blocks of four trees each, one tree per block representing each treatment. Air-layers allocated to each block were matched in size, and were pruned to six to eight leaves at planting. Trees were grown outdoors at the University of Florida, Tropical Research and Education Center (TREC) in Homestead, Florida (25.36◦N, 80.21◦W; 3 m above sea level). Mean ambient air temperatures in the area range from ca. 15◦ C in January to ca. 27◦ C in August (Getz 1979, NOAA 1999). Plants were spaced 0.8 m apart within and 1.5 m between rows. Pots were placed atop concrete blocks on a woven plastic ground cover to prevent contamination from the ground. Trees were irrigated with well water (pH 6.75) twice a day for 0.13 h (supplying ca. 15.6 l water per pot) by an automatic micro-irrigation system with one sprinkler in each pot to minimize splash. Irrigation with well water together with fertilization raised the pH (1:2 paste) of the potting medium to 7.09. Mycorrhizal fungus inoculum, of which we required 92 l, consisted of Bahia grass (Paspalum
87 notatum Flugge. var Pensacola) rhizomes and roots collected the day of planting from a field plot at TREC. Soil pH (1:2 paste) of the field plot was 7.02. Previous studies (Bakarr, 1997) have shown the grass roots to average 48.5% of their length colonized by mycorrhizal fungi, and the plot to contain 387 spores and 3.7 Glomus coremioides (Berk. et Broome) Redecker et Morton sporocarps per 50 g soil on average. Trapplants grown in the field soil have recovered as many as 14 species of Glomalean fungi, with species of the genus Glomus predominating. A ‘most probable number’ bioassay of soil from the plot indicated an average of 2122 mycorrhizal fungus propagules per 100 g soil (Bakarr, 1997). The inoculum-source plot was mown to the level of bare soil, above-ground litter was removed completely, and clumps of rhizomes and roots were collected by scraping the uppermost few centimeters depth of soil with a front-end loader. These clumps were shaken by hand to remove most soil, and rhizomes and roots were cut to 10–20 mm length by the swinging, weighted blades of a ‘hammer mill’ (Model JB-4 hammer mill, Jay-Bee Mfg., Tyler, TX). This mycorrhiza inoculum comprising rhizome pieces, root fragments, and adherent soil, was added to each +M pot (4.6 l pot−1 , n = 20) in a ca. 0.2 m diameter × 0.25 m deep hole in the center of the pot. The lychee air-layer root ball was embedded in the center of the inoculum, and the entire pot surface was covered with fumigated potting medium. For the control treatment, inoculum collected 13 days before the start of the experiment and fumigated the same way as the potting medium was added to each −M pot (4.6 l pot−1 , n = 20) in an identical fashion to the +M treatments. One-half litre of microbial filtrate prepared by soaking freshly collected grass rhizomes and roots in water for 3 days and then vacuum filtering the resulting infusion through Whatman No. 4 filter paper to exclude mycorrhizal fungi was added to each control pot on 2 February and again on 9 February 1998. Subsamples of fumigated potting medium, fumigated inoculum, and mycorrhizal fungus inoculum were collected, and concentrations of total nitrogen, nitrate nitrogen, total phosphorus, available phosphorous, and potassium were determined by the University of Florida, Analytical Research Laboratory (Gainesville, FL) (Table 1). Total nitrogen and phosphorus concentrations were determined after Kjeldahl digestion. Nitrate nitrogen, available phosphorous and potassium were extracted in de-ionized water as a Mehlich 1 saturated paste subsequently vacuum filtered
through Whatman No. 1 paper, after which nitrate nitrogen and phosphorus concentrations in the extract were determined by colorimetery in an Alpkem rapid-flow analyzer (Alpchem, College Station, TX) and potassium concentrations were determined by inductively coupled argon plasma spectroscopy (TJA Solutions, Franklin, MA) (Hanlon et al., 1994). Each pot received approximately 1 l of water with the following fertilizer composition per month: 36.72 g soluble 20-2-20 (Miller Chemical and Fertilizer, Hanover, PA), 0.15 g Sequestrene 138 Fe chelate (Ciba-Geigy, Greensboro, NC), and 0.1 ml Fer-a-gro Chelated Nutritional Spray (Florida East Coast Fertilizer, Homestead, FL). This constituted a ‘low P’ (LP) treatment (approximately 6 mg l−1 of P per pot per month). For a ‘high P’ (HP) treatment, in addition to the soluble low P fertilizer solution, a one-time addition of 124.9 g slow-release triple-superphosphate (Atlantic Fertilizer, Homestead, FL) was applied to each HP pot. The high-P level was approximately 134 mg l−1 of P per pot per month (assuming that the fertilizer releases for 9 months) which is what local growers typically apply to potted lychee trees propagated from air-layers. Additionally, 4 g of soluble ammonium nitrate was added to each pot on 13 April 1998, and 5 g of Sequestrene and 2 ml of Fera-gro were added to each pot on 3 and 17 August 1998, 28 September 1998, and 8 February 1999 to ameliorate nutrient deficiency symptoms. For reference, five air-layers that had been held for 1 month in 13.2 l pots of sterile potting medium were inoculated with a commercial inoculant (Accelerator Root Kit, Accelerator Horticultural Products, Port St. Lucie, FL; Table 2) on 2 March 1998. Approximately 1 kg of the commercial inoculant (containing at least 1700 spores of AM fungi; Table 2) was added to each of five 94.5 l black polyethylene pots to line the sides and bottom of a 0.2×0.25 m deep hole into which the root ball of the air-layer was subsequently placed. These pots were spaced 0.8 m apart supported on concrete blocks in a single row adjacent to those of the main experiment. Monthly soluble fertilizer additions were the same as for the LP treatment of the main experiment. Beginning 1 week after planting, the height of the tallest shoot apex, length of the longest leaflet, number of leaves, and stem diameter at a permanently marked position were measured. Measurements were made fortnightly until December 1998 and then at monthly intervals until May 1999. Although airlayers were matched approximately for size within
88 Table 1. Mean (n = 4) ± 1 SD mineral element concentrations of fumigated potting medium, fumigated field-collected mycorrhizal inoculum, and field-collected mycorrhizal inoculum Medium
TKN (µg g−1 )
NO3 -N (µg g−1 )
TP (µg g−1 )
P (µg g−1 )
Fumigated potting medium Fumigated mycorrhizal inoculum Mycorrhizal inoculum
5985±83
Not detectable
340±30
7423±290
0.05±0.09
2265±30
4.8±0.9
226.5±22.3
5378±196
Not detectable
1075±23
6.4±1.2
225.8±9.6
0.1±0
K (µg g−1 ) 74.4±5.0
TKN, total Kjeldahl nitrogen; TP, total phosphorus.
blocks at planting, they inevitably differed somewhat among blocks. In order to partially exclude this extraneous contribution to the variance among plants, for each variable the initial measurement was subtracted from each subsequent one before analysis by repeated-measures ANOVA. SYSTAT v. 5.01 (Wilkinson, 1990) was used for all statistical analyses with a probability level ≤ 0.05 determining significance. For plants in the main experiment, net CO2 assimilation of single leaves on all plants in each treatment was determined on 30 November 1998 (301 days after planting) with a LCA-3 portable gas analyzer (ADC, Hoddesdon, Herts., England) as described by Larson et al. (1991). The leaf cuvette of the gas analyzer was equipped with a portable lamp to maintain the photosynthetic photon flux above 1000 µmol m−2 s−1 , which is light saturating for net CO2 assimilation of lychee (Menzel and Simpson, 1994). Average air temperature in the cuvette during measurements was 31.8 ± 1.3◦ C (1 SD). Net CO2 assimilation was analyzed as a two-way ANOVA. Two or three mid-aged leaflets from each plant were collected on 30 November 1998 (10 months after planting) for nutrient analysis. Leaflet tissue was oven dried (at 70◦C for 3 days) and ground to a powder in a Wiley mill. Leaflet nutrient concentrations were determined at the University of Florida, Analytical Research Laboratory. Nitrogen concentrations were determined by the Total Kjedahl Nitrogen method. Portions of ground tissue samples were ashed in a muffle furnace, extracted in HCl, and Ca, Cu, Fe, K, Mg, Mn, P, and Zn concentrations were determined by inductively coupled argon plasma spectroscopy (TJA Solutions, Franklin, MA) (Hanlon et al., 1994). Indi-
vidual mineral element concentrations in leaflets were analyzed separately by ANOVA. Significance levels were Bonferroni corrected for the number of analyses performed (nine) by setting the critical probability level at 0.005 (∼0.05/9) (Scheiner, 1993). All plants were harvested on 17 and 18 May 1999. Leaves, shoots, root collars, and roots were collected separately and oven dried at 75 ◦ C to constant weight. Dry weights of plant organs were analyzed separately by two-way ANOVA. A representative subsample of fine roots (ca. 5% fresh weight) was removed from each root system for assessment of mycorrhizas after clearing and staining (Koske and Gemma, 1989). Proportion of root length colonized internally (usually by only hyphae and vesicles) was visually estimated (Giovannetti and Mosse, 1980) after dispersion of roots in a Petri dish and their examination under a dissecting microscope. Selected colonized root segments were mounted on slides for confirmation of their mycorrhizal status by examination with a compound microscope.
Results Plant survival was good throughout the experiment with just three plants (one −M HP and two +M HP) dying. Fine roots of all surviving control plants subsampled and stained for mycorrhizas at harvest, 469 days after planting, showed negligible colonization (n = 20, mean < 1%, range 0–5%) with just one plant having a colonization level (5%) similar to that of inoculated plants. That plant was not excluded from its group during subsequent growth analyses, making them conservative. Surviving inoculated plants
89 Table 2. Listed ingredients of the commercial mycorrhiza inoculum (Accelerator Root Kit, Accelerator Horticultural Products, Port St. Lucie, FL) used as a ‘reference’ inoculant Ingredient
Quantity, or source (for soil conditioners)
Live spores of arbuscular mycorrhizal fungi (includes Entrophospora columbiana, Glomus etunicatum, Glomus clarum, and Glomus sp.) Live spores of ectomycorrhizal fungi (includes Pisolithus tinctorius) Nitrogen fixing, phosphorus solubilizing, and growth promoting bacteria Humic acid (minimum 30% by weight) Complex carbohydrates and dried yeast Amino acids Yucca plant extract (wetting agent) Sea kelp extract (biostimulant) Water absorbent gel Nutrients (% by weight): N (3%), P (4%), K (3%), Ca (5%), S (2.8%), Mg (0.5%), Fe (0.4%)
Minimum of 300 spores of AM fungi per 6 oz scoop
had significantly higher levels of colonization than the controls (Kruskal–Wallis one-way ANOVA: Kruskal– Wallis statistic = 26.4, P < 0.001), although colonization was low with a range of 3–15% (n = 18, mean = 7.4%). Colonization of +M HP plants did not differ from that of +M LP plants (Kruskal–Wallis one-way ANOVA: Kruskal–Wallis statistic = 0.53, P = 0.467). Plants inoculated with the commercial inoculant formed AM with levels of colonization (n = 5, mean = 8.0%, range 5–10%) similar to those of all +M plants in the main experiment (Kruskal–Wallis one-way ANOVA: Kruskal–Wallis statistic = 0.58, P = 0.446). Although air-layers were originally matched for size within blocks, because mortality occurred in three different blocks we did not include a block effect in the analyses. Figure 1 shows the change from the initial measurement for the three morphological variables with a statistically significant between-subjects effect of mycorrhizal inoculation. Because two factor repeated measures ANOVA showed neither significant between-subjects effects of phosphorus fertilization nor of the interaction of inoculation and fertilization for any of the four morphological variables that we measured, data were reanalyzed as single-factor repeated measures ANOVA incorporating only the mycorrhiza inoculation factor. AM inoculation significantly affected between-subjects height change (F1,35 = 8.13, P = 0.007), leaflet length change (F1,35 =
Minimum of 40 million spores of P. tinctorius per 6 oz scoop Approximately 300 million combined per lb Natural humates Processed grain byproducts Animal and plant proteins Yucca schidigera Ascophylum nodosum Acrylamide copolymer gel Bone meal, blood meal, kelp meal, feather meal, sulphate of potash, langbeinite, humates, whey protein, rice bran, and rice hulls
6.46, P = 0.016), and change in number of leaves (F1,35 = 4.18, P = 0.048), but not change in stem diameter (F1,35 = 1.97, P = 0.170). Net CO2 assimilation determined on 30 November 1998 (301 days after planting) did not differ significantly among treatments (F1,36 = 0.00, P = 0.997). It averaged 4.1 ± 1.2 (1 SD) µmol CO2 m−2 s−1 (n = 38). Similar to the analyses of morphological growth measurements, two-way ANOVA performed on dry weights of plant parts after harvest found neither significant effects of phosphorus fertilization nor significant interaction of inoculation and fertilization for the dry weights of any plant part or for the total dry weights of whole plants. Mycorrhizal inoculation, however, significantly increased the dry weights of leaves (F1,33 = 6.67, P = 0.014), stems (F1,33 = 6.22, P = 0.018), and whole plants (F1,33 = 5.42, P = 0.026), but neither root collars (F1,33 = 1.79, P = 0.190) nor roots (F1,33 = 0.23, P = 0.634). Notwithstanding that inoculation did not affect root dry weight, the ratio of root dry weight to leaf dry weight was significantly diminished by inoculation (F1,33 = 12.09, P = 0.001), but neither by fertilization (F1,33 = 0.23, P = 0.636) nor the interaction of inoculation and fertilization (F1,33 = 1.13, P = 0.296). Reference plants inoculated with the commercial inoculant had a mean ratio of root to leaf dry weight identical to that of +M LP plants (0.206 for both groups versus 0.259 for −M LP plants). Figure 2 illustrates that the detectable ef-
90
Figure 2. Mean dry weights of Mauritius lychee trees at harvest, 469 days after inoculation with arbuscular mycorrhizal fungi (+M) or not (−M). Total weight of leaves and stems are shown above the x-axis, and total weight of root collars and roots are shown beneath the x-axis (as positive values). Probabilities (P) associated with F statistics for one-way ANOVAs for each plant part are shown; all n = 37.
Figure 1. Mean growth change (± 1 SE) from initial measurements of Mauritius lychee trees either inoculated (solid symbols, n = 18) with arbuscular mycorrhizal fungi or not (open symbols, n = 19). (A) Mean height from the surface of the substrate to the tallest shoot apex change. (B) Mean length of the longest leaflet change. (C) Mean number of leaves per plant change.
fects of inoculation on dry weight are manifested by above-ground parts (leaves and stems). Without regard to fertilization, the presence of AM resulted in a 41% leaf dry weight increase and 36% stem dry weight increase in comparison to plants lacking mycorrhizas 469 days after potting air-layers. Table 3 shows leaf tissue element concentrations for inoculated versus control plants (averaged across phosphorus treatments), reference commercial inoculant treated plants, and ranges of published mineral nutrient concentration standards (Menzel et al., 1992). Inoculated and control plants in the main experiment differed significantly (by Bonferroni-corrected one-
Figure 3. Mauritius lychee tree estimated mean total leaf element contents (= leaf dry weight at harvest × concentration at 301 days after planting) as a proportion of the highest value treatment (set equal to 1.0). Plants were inoculated with arbuscular mycorrhizal fungi (+M) or not (−M). NS, absolute element content not significantly different at P ≤ 0.05 after Bonferroni correction for the number of elements analyzed.
way ANOVA) for all element concentrations except total Kjeldahl nitrogen and iron (Table 3). Mycorrhizal plants had higher concentrations of phosphorus, potassium, copper, and zinc than control plants, but the latter had higher concentrations of calcium, magnesium, and manganese than the former (Table 3). When total leaf element contents were estimated as the product of concentration and leaf dry weight at harvest, however, mycorrhizal plants significantly exceeded control plants for all elements except calcium, magnesium, and manganese which did not differ significantly (Fig. 3). Leaf tissue element concentrations did not differ between plants inoculated with fieldcollected roots versus the commercial inoculant except in the case of iron which was 1.75 times more concen-
91 Table 3. Air-layered Mauritius lychee leaf mean ± 1 SD element concentrations 301 days after planting of control plants (−M; n = 19), arbuscular mycorrhizal plants (+M; n = 18), and reference plants inoculated with a commercial inoculant (REF; n = 5). One-way ANOVA results compare −M versus +M plants (n = 37) and +M versus REF plants (n = 23), respectively. Minimum and maximum leaf nutrient concentration standards (STD) developed for lychee in South Africa, Israel, and Australia (Menzel et al., 1992) are shown. Boldface indicates element concentrations below minimum standards
−M +M F1,35 P REF F1,21 P STD
TKN (mg g−1 )
P (mg g−1 )
K (mg g−1 )
Ca (mg g−1 )
Mg (mg g−1 )
Mn (µg g−1 )
Cu (µg g−1 )
Zn (µg g−1 )
Fe (µg g−1 )
20.73±3.21 22.58±2.79 3.47 0.071 21.14±2.60 1.06 0.314 13.0–18.0
1.87±0.35 2.30±0.32 15.04
