Annals of Botany 84 : 771780, 1999 Article No. anbo.1999.0977, available online at http:\\www.idealibrary.

com on

Inuence of Flooding on Net CO2 Assimilation, Growth and Stem Anatomy of Annona Species
! N 4 E Z -E L I S E A*, B R U C E S C H A F F ER*, J A C K B. F I S H E R, ROBERTO NU A N G E L M. C O L L S* and J O N A T H A N H. C R A N E* * Uni ersity of Florida, IFAS, Tropical Research & Education Center, 18905 SW 280th Street, Homestead, FL 33031, USA and Fairchild Tropical Garden, 11935 Old Cutler Rd., Miami, FL 33156, USA and Department of Biological Sciences, Florida International Uni ersity, Miami, FL 33199, USA
Received : 8 June 1999 Returned for revision : 20 July 1999 Accepted : 23 August 1999

A series of experiments was conducted to assess net CO assimilation and growth responses to waterlogging of grafted # and seedling trees in the genus Annona. Seedlings of A. glabra, A. muricata and A. squamosa L., and scions of Gefner atemoya (A. squamosaiA. cherimola Mill.), 49-11 ( Gefner atemoyaiA. reticulata L.), 4-5 ( Priestley atemoyaiA. reticulata), A. reticulata grafted onto either A. glabra, A. reticulata or A. squamosa rootstocks were ooded for up to 60 d. Soil anaerobiosis occurred on the third day of ooding. Seedlings of A. glabra and A. muricata, and the scions 49-11 , Gefner atemoya, and A. reticulata grafted onto A. glabra rootstock were considered ood tolerant based on their ability to survive and grow in ooded conditions. Scions of the normally ood-sensitive A. reticulata, Gefner atemoya, and 49-11 tolerated root waterlogging when grafted onto the ood-tolerant species, A. glabra. In contrast, ooding of A. squamosa seedlings and rootstocks, and A. reticulata rootstocks greatly reduced growth and net CO assimilation rates, and resulted in 2080 % tree mortality. Stem anatomical responses to long# term ooding (12 continuous months) were assessed in seedlings of A. glabra and A. muricata, and trees of 49-11 grafted onto A. glabra. Flooded trees developed hypertrophied stem lenticels, particularly in A. glabra, and enlarged xylem cells resulting in thicker stems with reduced xylem density. Flooding did not increase air spaces in pre-existing xylem near the pith or in xylem tissue that was formed during ooding. Thus, ood tolerance did not involve aerenchyma formation in the stem. # 1999 Annals of Botany Company Key words : Flood tolerance, net CO assimilation, photosynthesis, stem anatomy, shoot growth, anaerobiosis, # Annonaceae.

I N T R O D U C T I ON Urban development is gradually displacing fruit production in several tropical and subtropical parts of the world to areas with soils that are poorly drained or are exposed to recurrent ooding. An ecient use of agricultural areas subjected to periodic waterlogging will require identication of ood-tolerant crops, including fruit trees, with economic potential (Schaer, 1998). The genus Annona includes several fruit trees of economic importance in temperate, subtropical, and tropical areas throughout the world (Popenoe, 1920 ; Morton, 1987). In some subtropical areas, sugar apple (Annona squamosa L.) and atemoya (A. squamosa L.iA. cherimola Mill.) are grown commercially, while soursop (A. muricata L.), the most tropical species in the genus Annona, is grown occasionally as a door-yard tree. A non-commercial species of Annona native to the tropical Americas, pond apple (A. glabra L.) is reported to be well adapted to ooded conditions (Noonan, 1953 ; Morton, 1987 ; Nakasone and Paull, 1998). This species is graft compatible with several commercial Annona species (Sanewski et al., 1991), and it has been suggested that its

For correspondence. E-mail bas!gnv.ifas.u.edu

use as a rootstock could allow commercial, ood-sensitive Annona species to be grown in areas subjected to periodic ooding (Popenoe, 1920 ; Kennard and Winters, 1960). Flood tolerance is determined by the ability of a plant to grow and survive in soils with a water content above eld capacity (Rowe and Beardsell, 1973). Soil ooding disrupts the metabolism of mesophytic plant species by displacing O # from soil pores and promoting O depletion by roots and # soil microbes (Drew, 1990). Plant responses to ooding can include stomatal closure, premature leaf senescence, reduced root and shoot growth, formation of adventitious roots, aerenchyma, and hypertrophied lenticels (Kozlowski, 1997). Stomatal closure can be particularly critical since it often reduces carbon assimilation at moderate to high light intensities (Kozlowski, 1997). There is a general lack of information on the anatomical, physiological and growth responses of Annona species to ooding. Such information is needed to understand better the adaptive mechanisms employed by ood-tolerant Annona species, such as A. glabra, and may eventually aid the development of ood-tolerant species and\or cultivars. This study was conducted to determine the eects of ooding on plant survival, growth, stem anatomy and net CO as# similation of several species and cultivars of seedling and grafted trees in the genus Annona. # 1999 Annals of Botany Company

0305-7364\99\120771j10 $30.00\0

772

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

MATERIALS AND METHODS platinum-tipped microelectrodes, as described by Crane and Davies (1988). Fifteen continuously-ooded plants and 15 plants subjected to cyclical ooding (three each of the ve tree types for each of the two ooding methods) were monitored for changes in soil Eh. Plant growth. Immediately before treatments were imposed, trees were 0n91n5 m tall and consisted of veeight lateral branches. Annona trees are semi-deciduous, shedding leaves during the spring under subtropical conditions. A unique botanical feature is that buds are often inserted within leaf petioles, causing them to remain dormant while leaves remain attached (Sanewski, 1991). New, uniform vegetative shoots were produced for growth and leaf production measurements by clipping a lateral branch of each plant (1015 cm were removed with pruning shears) and cutting o the distal four leaves on day 0. The released buds were swollen within 7 d of removing leaves, indicating emergence of new shoots. Thus, the pruning and defoliation procedure was useful in eliminating the inhibitory eect of the presence of leaves as a source of experimental error. One new shoot was chosen per tree on which shoot length and leaf number were recorded 14 and 50 d after initiating ooding treatments. Basal trunk diameter of each plant was recorded 10 cm above the soil surface on days 0, 14 and 50. Net CO assimilation. Net CO assimilation (A) was # # measured periodically for all plants. Immediately before treatments were imposed, several leaves of the same physiological age (recently fully expanded) were chosen for gas exchange determinations. A was measured on these leaves throughout the experiment, although a dierent leaf from the group was selected on each measurement date. This was done to ensure a sucient number of uniform leaves for photosynthesis measurements, as no new vegetative shoots are produced while leaves are still attached to trees (George and Nissen, 1991 ; Sanewski, 1991). Selected leaves were located on the pruned branch, basal to the newly-formed vegetative shoots used for growth measurements. Selecting leaves from the pruned branch also helped to reduce possible variation among leaves due to their position within the canopy. A portable infrared gas analyser (LCA-3, Analytical Development Co., Hoddesdon, Herts., UK) was used for measurements as described by Larson et al., 1991 b). The rate of air ow into the leaf chamber was 400 ml min". Measurements were made between 1200 and 1500 h at a photosynthetic photon ux density 1000 mol m# s". Experiment 2. Eect of ooding on three dierent scion\rootstock combinations Glasshouse and plant material. The scion\rootstock combinations 4-5 \A. reticulata, Gefner atemoya\A. squamosa, and A. reticulata\A. glabra were compared for tolerance to ooding under glasshouse conditions as described for expt 1. The 4-5 hybrid was developed by Zill High Performance Plants (Boynton Beach, Florida, USA). Trees were planted in 12 l plastic containers in Krome very gravelly loam soil. Flooding method, experimental design and measurements. Trees of each of the three scion\rootstock combinations

Plant material Seven Annona species were used throughout this study either as seedling trees or in dierent scion\rootstock combinations, as detailed below for each of the four separate experiments. The tested species were Annona glabra L. (pond apple), A. muricata L. (soursop or guana! bana), A. squamosa L. (sugar apple), and A. reticulata L. (bullocks heart), as well as the hybrids A. cherimola L.iA. squamosa L. (atemoya), Priestley atemoyaiA. reticulata ( 4-5 ), and Gefner atemoyaiA. reticulata L. ( 49-11 ). The hybrids 4-5 and 49-11 were developed by Zill High Performance Plants (Boynton Beach, Florida, USA) and are currently under evaluation. All trees used in the study were 2 to 3 years old. Experiment 1. Impact of ooding on three species of Annona seedling trees and comparison of two dierent rootstocks Glasshouse and plant material. The study was conducted during JuneAugust 1997 in a glasshouse at the University of Florida, Tropical Research and Education Center in Homestead, USA. Average temperatures during the experimental period were maintained at 3235 mC during the day and 2630 mC during the night ; relative humidity was maintained at 75 %. Flooding eects were measured on ve types of trees. Seedling and grafted trees between 2 and 3 years of age were used. Seedling trees tested were A. glabra, A. muricata and A. squamosa, whereas grafted trees consisted of 49-11 \A. glabra ( 49-11 scion grafted onto A. glabra rootstock) and 49-11 \A. reticulata. All plants were grown in 20 l plastic containers in Krome very gravelly loam soil (loamy-skeletal, carbonatic, hyperthermic Lithic Rendoll) which is native to the agricultural area in southern Florida near Everglades National Park (Noble et al., 1996). Flooding method and experimental design. Plants were subjected to continuous ooding for a period of 50 d or to three ooding cycles at 10 d intervals, from days 0 to 10, 20 to 30, and 40 to 50, during a 50 d period. Plants ooded in a cyclical manner were irrigated daily between ooding episodes, from days 10 to 20 and 30 to 40. Flooding was imposed by placing the potted trees in 35 l plastic containers and adding tap water to approx. 5 cm above the soil surface. Water was added daily to replace loss due to evapotranspiration. Non-ooded (control) plants were irrigated daily to maintain soil moisture near container capacity. Treatments were arranged in a randomized complete block design, with ve single-plant replicates of each plant type per treatment. Soil redox potential. Anaerobic (reduced) conditions occur at soil redox potentials (Eh) 200 mV (Ponnamperuna, 1984). Eh of continuously and cyclically ooded soil was monitored periodically during the 50 d ooding period to detect the transition from aerobic (oxidized) to anaerobic conditions in the plant rhizosphere. Soil Eh was measured at a soil depth of 20 cm using Ag+\AgCl reference electrodes (Model RC5, Jensen Instruments, Tacoma WA, USA), an oxygen meter (Model P5E, Jensen Instruments) and

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

were either subjected to continuous ooding for 30 d as described for the previous experiment, or not ooded and irrigated daily (controls). A ooding period of 30 d was chosen because the previous experiment showed that A. reticulata and A. squamosa rootstocks were susceptible to ooding. Net CO assimilation was measured 2 and 4 weeks # after termination of the ooding period to assess whether photosynthetic capacity could be recovered. The experimental design was a randomized complete block with six treatments, and ve single-tree replicates per treatment. Growth measurements included shoot length, the number of leaves per shoot, and stem diameter. Measurements on all trees were made 0, 17, 31 and 59 d after ooding was imposed, and 1 and 29 d after ooding was discontinued in the ooding treatment. A branch of each tree was pruned 4 weeks before starting the experiment to generate a uniform population of new vegetative shoots for growth measurements. One individual new vegetative shoot from the pruned branch was selected per tree to record shoot growth and leaf production. Trunk diameter, 10 cm above soil surface, was recorded on days 0 and 59. Net CO assimilation was measured periodically during # 58 d as described for expt 1, beginning on the day that ooding was initiated (day 0). Experiment 3. Flood tolerance of Gefner atemoya grafted onto A. squamosa and A. glabra rootstocks Plant material. The scion\rootstock combinations of Gefner atemoya\A. glabra, and Gefner atemoya\A. squamosa were tested for tolerance to ooding under outdoor conditions during the summer of 1998. Since Gefner atemoya is graft-incompatible with A. glabra (Sanewski, 1991), incompatibility was overcome by using an intergraft of 49-11 , which is compatible with both atemoya and A. glabra. A dierent potting medium was used in this experiment to determine whether ooding tolerance of A. squamosa and A. glabra rootstock was modied when plants were grown in a dierent soil. Trees were planted in 12 l plastic containers in peat, cypress\hardwood chips, and sand (50 : 30 : 20 by volume), and slow-release fertilizer (13N-13P O -13K O, # & # 4n5 kg m$). All trees were irrigated daily by overhead sprinklers for 1 h (from 1430 to 1530 h) during the experiment. Flooding method, experimental design and measurements. Flooding was imposed as described for expt 1. Trees of each scion\rootstock combination were ooded continuously for 30 d or not ooded but irrigated daily (controls). Thus, the experiment consisted of two ooding treatments and two scion\rootstock combinations per treatment in a randomized complete block design, with each treatment consisting of six single-tree replicates for each scion\rootstock combination. One branch was pruned on each tree 4 weeks before imposing treatments to generate a uniform population of vegetative shoots. An individual vegetative shoot was selected from each pruned branch to record shoot growth and leaf production per tree. Trunk diameter was measured on days 0 and 62. Net CO assimilation was measured as # described for expt 1. Data were collected periodically for

773

62 d after starting treatments, beginning on the day that ooding was initiated (day 0). Experiment 4. Eect of long-term ooding on the stem anatomy of A. glabra and A. muricata Trees of A. glabra, A. muricata, and 49-11 \A. glabra were ooded continuously for 12 months or not ooded and examined for changes in stem anatomy. Three ooded and three non-ooded plants for each tree type were used. After 12 months, a stem sample was taken from the base of each plant, 25 cm immediately distal to the upper-most root and at the water line. The transverse width of pith, xylem and bark was measured on ve radii for each sample. Stem samples were split longitudinally through the pith. One thick slice of peripheral xylem was weighed for each stem, its fresh volume determined by water displacement, and weighed after drying for 5 d in an oven at 70 mC. The remaining ve split segments were xed in formaldehydealcohol-acetic acid and processed by washing in water and alcohol, sectioning on a sliding microtome at about 3050 m, adhering the sections to slides, staining with safranin and fast green, and mounting permanently under a cover glass. Cell size was measured directly with an ocular micrometer on a compound microscope. Data analysis Data in all experiments were analysed by analysis of variance (ANOVA) and Tukeys studentized multiple range tests, and\or standard t-tests using SAS software for personal computers (SAS Institute, 1996). RESULTS Experiment 1. Impact of ooding on grafted and seedling Annona species Soil redox potential (Eh). There were no dierences in soil Eh among the ve dierent Annona species or cultivars

600 Soil redox potential (mV) aerobic 400

200

0 anaerobic 200 0 10 20 30 40 50 Days after imposition of flooding

F. 1. Soil redox potential (Eh) of Rockdale limestone soil subjected to cyclic (days 010, 2030 and 4050) ($) and continuous (50 d) (#) ooding. Anaerobic (reduced) conditions occur at Eh 200 mV. Symbols represent meansps.e. of 15 container-grown plants. Standard error (s.e.) bars are smaller than the symbols for the means.

774

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

T 1. Shoot growth, owering and stem diameter of container-grown seedling and grafted Annona trees in response to cyclic and continuous ooding under glasshouse conditions. A branch was pruned on each tree on day 0 and one egetati e shoot was selected from each pruned branch to record shoot growth and leaf production
Shoot length (cm) Flooding treatments 49-11 \A. glabra Non-ooded* Cyclically ooded Continuously ooded 49-11 \A. reticulata Non-ooded* Cyclically ooded Continuously ooded A. glabra Non-ooded* Cyclically ooded Continuously ooded A. muricata Non-ooded* Cyclically ooded Continuously ooded A. squamosa Non-ooded* Cyclically ooded Continuously ooded * test Day 14 Day 50 No. of leaves per shoot Day 14 Day 50 Day 0 Stem diameter (cm) Day 14 Day 50

1n7b 3n8a 4n7a 2n6a 1n8b 1n5b 3n4a 2n5a 3n6a 5n3a 3n9ab 3n1b 2n9a 1n2b 1n1b

4n8c 10n5b 18n0a 9n6a 4n7b 5n3b 12n6a 7n9b 12n1a 14n0a 8n1b 6n9b 5n4a 2n9b 1n9b

0n8b 2n8a 2n9a 2n0a 1n6a 1n2a 2n2a 1n9a 2n6a 3n2a 2n6a 2n4a 2n4a 0n8b 0n9b

4n7b 9n6a 8n8ab 6n6a 4n7b 1n3c 6n9a 6n6a 5n6a 9n0a 6n2a 7n1a 4n3a 3n9a 4n5a

4n6a 4n1a 4n4a 4n2a 4n5a 4n5a 3n9a 4n3a 4n2a 3n4a 3n1a 3n5a 3n2a 3n3a 3n6a

4n7a 4n3a 4n5a 4n2a 4n6a 4n7a 4n1b 4n5a 4n3ab 3n5a 3n1a 3n6a 3n2a 3n4a 3n6a

5n1b 6n3ab 6n9a 4n4a 4n8a 4n9a 4n2b 5n6a 6n1a 3n7a 3n3a 3n7a 3n4a 3n5a 3n7a

Control trees were not ooded, but were irrigated daily to container capacity. Flooding was imposed on days 010, 2030 and 4050. Trees were irrigated as controls on days 1020 and 3040. Trees were ooded for 50 continuous days. Means followed by dierent superscripts indicate signicant dierences among ooding treatments according to Tukeys studentized range (P 0n05).

T 2. Shoot growth and stem diameter of container-grown Annona trees of three scion\rootstock combinations in response to cyclic and continuous ooding under glasshouse conditions. A branch was pruned on each tree 4 weeks before commencing the experiment. One egetati e shoot was selected from each pruned branch to record shoot growth and leaf production
Shoot length (cm) Flooding treatment A. reticulata\A. glabra Non-ooded* Flooded Signicance Gefner \A. squamosa Non-ooded* Flooded Signicance 4-5 \A. reticulata Non-ooded* Flooded Signicance Day 0 Day 17 Day 31 Day 59 Day 0 No. of leaves\shoot Day 17 Day 31 Day 59

1n7 1n9 ns 1n8 2n2 ns 1n9 1n6 ns

12n1 12n4 ns 10n1 6n3 ** 11n2 7n0 **

19n0 21n6 ** 23n9 9n5 ** 20n7 7n4 ***

28n6 29n5 ns 33n8 11n8 *** 30n0 11n8 ***

1n2 1n2 ns 1n2 1n6 ns 1n0 1n0 ns

3n8 4n4 * 4n4 2n4 *** 3n8 3n2 *

7n2 9n4 *** 7n3 4n0 *** 7n8 4n6 ***

14n8 14n8 ns 13n8 5n5 *** 12n4 8n0 ***

* Control trees were not ooded, but were irrigated daily to container capacity. Trees were ooded for 30 continuous days. ***, **, and * indicate signicant dierence between ooding treatments at P 0n01, 0n05 and 0n1, respectively, and ns indicates no signicant dierence according to standard t-tests.

(data not shown), therefore data from the 15 plants of each ooding treatment were pooled. For continuously-ooded trees, the initial Eh (day 0) was 423 mV, indicating an aerobic root environment (Fig. 1). Anaerobic conditions below Eh 200 mV occurred on the third day of ooding (196 mV), and Eh decreased gradually until day 15 (Fig. 1).

Average Eh values changed little after day 20, remaining at about k155 mV until the end of the experimental period. Anaerobiosis of cyclically-ooded soil was reached on day 3 (Eh l 164 mV). On day 10 (immediately prior to unooding trees), Eh reached 87 mV but increased to 387 mV (aerobic conditions) by day 15. Soil was aerobic on

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

10 8 6 4 2 0 10 8 '49-11'/A. glabra non-flooded cyclically flooded continuously flooded 4 3 2 1 0 'Gefner' atemoya/A. squamosa 5 '49-11'/A. reticulata 5 A. reticulata / A. glabra

775

A (mol CO2 m2 s1)

non-flooded flooded

A (mol CO2 m2 s1)

6 4 2 0 0 flooded flooded flooded 50

4 3 2 1 0 '4-5'/A. reticulata 5 4 3 2 1 0

10 20 30 40 Days after imposition of flooding

F. 2. Eect of cyclic (days 010, 2030 and 4050) and continuous (50 d) ooding on net CO assimilation (A) of the hybrid 49-11 # grafted onto two dierent rootstocks. Symbols represent means of ve treesps.e.

flooded

flooded

flooded non-flooded cyclically flooded continuously flooded

A (mol CO2 m2 s1)

4 3 2 1 0 1 0

10

50 20 30 40 Days after imposition of flooding

60

10 20 30 40 50 Days after imposition of initial flooding

F. 4. Eect of continuous ooding (30 d) on net CO assimilation (A) # of three Annona scion\rootstock combinations. Symbols represent means of ve treesps.e.

F. 3. Eect of cyclic (days 010, 2030 and 4050) and continuous (50 d) ooding on net CO assimilation (A) of Annona squamosa # seedling trees. Symbols represent means of six treesps.e.

days 15 and 20, and anaerobic again on day 25, after a further 5 d of ooding. The aerobic-anaerobic cycle was repeated on days 40 and 50 as a result of the nal ooding cycle imposed on days 4050 (Fig. 1). Growth. Shoot length, leaf production and stem growth were stimulated by ooding in 49-11 \A. glabra, whereas cyclic and continuous ooding reduced shoot extension of 49-11 \A. reticulata by about 50 % (Table 1). Cyclic ooding reduced leaf production by about 30 % and continuous ooding by 80 %. The enhanced growth of

shoots from 49-11 \A. glabra trees exposed to either cyclic or continuous ooding was already apparent on day 14, when shoot length and leaf number of ooded trees was at least double that of non-ooded controls. Shoot extension and leaf production increased sharply during the following 5 weeks, and by the nal measurement on day 50, shoots of ooded 49-11 \A. glabra trees were nearly twice (cyclic ooding) to more than three-times (continuous ooding) the length of controls. Shoot growth of A. muricata and A. squamosa was inhibited by both cyclic and continuous ooding (Table 2). However, there was no signicant eect of either cyclic or continuous ooding on nal leaf production of these two species. Stem diameter of all trees was generally unaected

776

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

T 3. Growth responses of container-grown Gefner atemoya (A. cherimolaiA. squamosa) grafted onto two dierent rootstocks, to continuous ooding outdoors. A branch from each tree was pruned 4 weeks before imposing treatments to generate new egetati e shoots. One egetati e shoot was selected from each pruned branch to record shoot growth and leaf production
Shoot length (cm) Flooding treatments Gefner \A. squamosa Non-ooded Flooded Signicance Gefner \A. glabra* Non-ooded Flooded Signicance Day 0 Day 16 Day 33 Day 62 Day 0 No. of leaves per shoot Day 16 Day 33 Day 62

2n3 2n2 ns 1n8 2n4 ns

14n4 8n7 ** 10n7 14n3 *

31n4 20n8 * 22n0 23n9 ns

37n6 25n4 32n1 34n2 ns

1n5 ns 0n8 1n8 **

4n0 3n2 ** 3n5 3n2 ns

11n8 8n0 * 9n0 10n0 ns

15n5 10n0 15n7 14n8 ns

* Gefner atemoya is graft-incompatible with A. glabra ; it was grafted onto A. glabra using 49-11 interstock. Trees were ooded for 30 continuous days. ***, **, and * indicate signicant dierence between ooding treatments at P 0n01, 0n05 and 0n1, respectively, and ns indicates no signicant dierence according to standard t-tests. Data from only one tree (rep) ; remaining trees died as a result of ooding stress. Therefore, mean separation could not be done.

by ooding treatments for the rst 14 d of the experiment (Table 1). Stem diameter of ooded A. glabra and 4911 \A. glabra trees increased by 3057 % from day 0 to day 50, but stem diameter of 49-11 \A. reticulata, A. muricata and A. squamosa trees was unaected by ooding (Table 1). Net CO assimilation. Net CO assimilation rates of # # ooded and non-ooded trees declined over time due to leaf aging owing to the semi-deciduous nature of Annona species (George and Nissen, 1991). It was necessary to follow A on leaves of the same age throughout the ooding period because ooding can aect the timing of budbreak (Schaer et al., 1992). The mean relative decline was generally greater for ooded than non-ooded trees (Figs 2 and 3). However, imposing cyclic or continuous ooding had no signicant eect on A rates for 49-11 \A. glabra or A. glabra seedlings compared to those of controls. Similarly, seedling trees of A. glabra subjected to three 10 d ooding cycles or to continuous ooding for 50 d had A rates comparable to those of controls and of A. muricata (Fig. 3). In contrast, trees of 49-11 \A. reticulata and A. squamosa had signicant reductions (generally 50 %) of A compared to controls (Fig. 2). The eect was greater for trees under continuous ooding than for those exposed to cyclic ooding, as trees were not assimilating CO , but respiring, # on days 10, 40 and 50 (Fig. 2). Experiment 2. Eect of ooding on three dierent scion\rootstock combinations Growth. Flooding for 30 d did not reduce shoot extension or leaf production in trees of A. reticulata\A. glabra in this experiment (Table 2). Flooded trees of Gefner \A. squamosa showed reduced shoot length and leaf production as of day 17 (60 and 83 %, respectively). Flooded trees of 45 \A. reticulata also showed a reduction in shoot extension and leaf production from day 17 (60 and 69 %, respectively). Three 4-5 \A. reticulata (60 %) and two Gefner \A. squamosa (40 %) trees died 39 and 51 d after

starting treatments (data not shown). Mean shoot growth and leaf production was calculated from living trees only. Trunk diameter of all control treatments and ooded trees of 4-5 \A. reticulata and Gefner \A. squamosa increased by 3 to 10 % from day 0 to day 59, and by 16 % for ooded trees of A. reticulata\A. glabra (data not shown). Net CO assimilation. There were no reductions (except # for day 45) in A for ooded trees of A. reticulata\A. glabra in relation to controls (Fig. 4). However, reductions in A were recorded for ooded trees of both Gefner \A. squamosa and 4-5 \A. reticulata, particularly after 5 d of ooding (Fig. 4). Experiment 3. Flood tolerance of Gefner atemoya grafted onto A. squamosa and A. glabra Growth. Flooding reduced shoot extension and leaf production of Gefner \A. squamosa (day 16), but, more signicantly, it caused the death of 60 % of these trees by day 33 and 80 % by day 62 (Table 3). Shoot growth and leaf production on trees of Gefner \A. glabra were not aected by 30 or 60 d of continuous ooding. Trunk diameter increased by 16n7 % from day 0 to day 62 for ooded trees of Gefner \A. glabra, and by 3n26n8 % for all other ooded trees (data not shown). Net CO assimilation. Compared to control trees, Gefner # atemoya tolerated ooding when grafted onto A. glabra, but not when grafted onto A. squamosa. In relation to day 0, there was a reduction of approx. 60 % in A for all trees grafted onto A. glabra rootstock, including controls, during the nal 2 weeks of the experiment (data not shown). Experiment 4. Eect of long-term ooding on stem anatomy of A. glabra and A. muricata Stem structure. Flooding increased total stem xylem radius and bre cell radius, and reduced stem xylem density of A. glabra and 49-11 \A. glabra, but not of A. muricata.

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

8 non-flooded flooded for 30 days

777

A (mol CO2 m2 s1)

0 0 10 20 30 40 50 Days after imposition of flooding 60

F. 5. Eect of continuous ooding (30 d) on net CO assimilation (A) # of Gefner atemoya grafted onto A. squamosa. Symbols represent means of ve treesps.e.

The proportional increase in xylem radius is fully accounted for by increased bre cell radius. Therefore, the activity of the vascular cambium (i.e. the radial number of bres) was unaected by ooding. The wood of A. glabra and A. muricata was relatively soft and light weight. The density of A. glabra was about half that of A. muricata (0n165 s. 0n398 g cm$ ; Table 4). In both species, the xylem had many thin-walled bres and groups of onethree, rarely fourve, narrow vessels (Fig. 6). The greater density of A. muricata xylem was due to the presence of thicker bre walls, which were more lignied than those of A. glabra, as indicated by increased safranin staining. The xylem rays were onethree cells wide in A. glabra and broader in A. muricata, often veseven cells at their greatest tangential width. In all respects, stem wood of the A. glabra rootstock (in trees of 49-11 \A. glabra) was similar to that of A. glabra seedling trees (Table 4), and therefore is not shown in Fig. 6. There was no evidence of increased air spaces in xylem tissues of ooded stems. The bark of A. glabra and A. muricata was typically thin and brous under non-ooded conditions. Bark thickness was unaected by ooding (Table 4) and there was no increase of intercellular air spaces in the secondary phloem and cortex of ooded stems. Neither A. glabra nor A. muricata showed evidence of aerenchyma in

F. 6. Eect of ooding on stem anatomy of A. muricata (A, B) and A. glabra (C, D). Xylem rays (darkened vertical bands for both species) were veseven cells wide in A. muricata and onethree cells wide in A. glabra. Fibres and vessels of ooded stems (B, D) had thinner walls and were less lignied than those of non-ooded stems (A, C), particularly for A. glabra. Asterisks indicate radial row of bres measured. Note absence of new air canals or intercellular spaces for ooded stems of both species. All at same magnication ; bar l 200 m.

T 4. Anatomical features of ooded and non-ooded trees of Annona glabra, A. muricata, and 49-11 \A. glabra. Flooding was imposed for 12 continuous months in a glasshouse
A. glabra Anatomical feature Stem radius (mm) Bark radius (mm) Xylem radius (mm) Fibre cell radius ( m) Xylem density (g cm$) Flooded 36n8 2n5 33n4 46n4 0n165 Nonooded 23n0 2n4 18n9 28n3 0n290 Signicance *** ns *** ** *** Flooded 18n8 2n3 15n4 23n9 0n398 A. muricata Nonooded 17n2 2n1 13n9 22n9 0n444 Signicance ns ns ns ns ns 49-11 \A. glabra Flooded 29n4 2n0 27n3 46n2 0n114 Nonooded 19n0 2n1 14n7 37n6 0n242 Signicance *** ns *** * **

Data are means of three trees per species. ***, **, and * indicate signicant dierences between ooded and non-ooded trees at P no signicant dierences according to standard t-tests.

0n01, 0n05 and 0n1, respectively, and ns indicates

778

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

A. glabra as a rootstock and the susceptibility of A. squamosa to ooding. The results indicate that Annona species grafted onto A. glabra rootstock, as well as trees of A. muricata, may tolerate prolonged ooding episodes in natural wetlands or in ood-prone agricultural areas. The general eect of ooding on photosynthesis throughout this study was a reduction in A rates. However, reductions in A over time were often found for non-ooded trees. This was probably due to the aging of leaves during the course of the experiments. Net CO measurements were # made initially on 8- to 12-week-old leaves located on a single branch. It was necessary to monitor A on the same group of leaves over time, as no synchronous leaf production occurs on Annona trees until they defoliate (George and Nissen, 1991). The reduced A rates as a result of ooding were particularly pronounced for Gefner \A. squamosa, 4-5 \ A. reticulata, 49-11 \A. reticulata and A. squamosa seedlings. For Gefner \A. squamosa, photosynthetic rates declined drastically within the rst week of ooding, and remained below 1n0 mol CO m# s" after day 20. The # reduction was probably due to the declining condition of trees, since 60 % of trees (three plants) died around day 30. It is reported that Gefner atemoya seedlings or trees grafted onto atemoya, A. squamosa, or A. reticulata rootstock are susceptible to ooding under eld conditions (Campbell and Phillips, 1994). Growth and photosynthesis results of expt 3 suggest that trees of Gefner atemoya intergrafted (with 49-11 ) onto A. glabra rootstock may be productive in areas with episodic ooding. Trunk diameter of A. glabra seedlings or rootstocks ( 4911 \A. glabra), but not of A. muricata, was greater in ooded than non-ooded soil (Table 4). Anatomical examination showed that bark thickness was not aected, but xylem thickness, bre radial width, and xylem density were signicantly higher in ooded plants compared to nonooded plants. In A. glabra, the ratio of ooded to nonooded xylem width was 33n4\18n9 l 1n767 and the ratio for bre radial width was 1n639. Since bre cells make up most of the xylem, these results indicate that the increased xylem width resulted from an increase in bre cell size, rather than an increase in bre number caused by increased activity of the vascular cambium. Additionally, the decrease in xylem density was caused by an increase in bre cell size, and not an increase in cell wall material. Fibres and vessels in the xylem of ooded stems seemed to have thinner and less lignied walls compared to those of non-ooded stems, although these features were not quantied in this study. In trees of 49-11 \A. glabra, stems were similar to those of non-grafted (seedling) A. glabra trees. A similar but less pronounced response was seen in xylem bres and vessels of A. muricata stems subjected to long-term ooding. No well-developed intercellular air spaces were observed in the stem xylem and phloem of any ooded plants. We expected to nd aerenchyma tissue with large intercellular air spaces (Fahn, 1990), present in the swollen stem base because this is the boundary, at the water line, between the aerobic shoot and the submerged stem base and root system. Such aerenchyma could have facilitated gas exchange between the shoot and the submerged parts of the tree. Aerenchyma formation is well-documented in primary

their non-ooded and ooded stems in the region at or just above the ood water line. D I S C U S S I ON Flooding resulted in anaerobic soil conditions within 3 d of imposing treatments, as indicated by soil Eh values (Fig. 1). The results were comparable to those observed previously for mango trees ooded in the same type of soil (Larson et al., 1991 a, b). Shoot extension and leaf production of seedling and grafted trees generally decreased in response to ooding (Tables 24), except for trees of 49-11 \A. glabra. The nding that ooding enhanced shoot growth of 4911 \A. glabra appeared unusual, since ooding typically results in decreased growth rates (Schaer et al., 1992 ; Kozlowski, 1997). In preliminary trials we also observed that ooded trees of 49-11 \A. glabra had greater shoot growth than non-ooded ones (data not shown). Flooding generally suppresses growth in both angiosperms and gymnosperms (Kozlowski, 1997). However, in seedlings of Pinus halepensis, short-term ooding (10 d) stimulated formation of secondary needles, while longer ooding periods arrested their formation (Sena Gomes and Kozlowski, 1980). For seedling trees of A. glabra in our study, all treatments caused similar shoot extension and leaf production. It is not clear why shoot growth of A. glabra seedlings was not stimulated by ooding as was the case for A. glabra when used as a rootstock for 49-11 (Table 1). Net CO assimilation rates were similar for these two types # of trees (Figs 2 and 3). Therefore, it is possible that in trees of 49-11 \A. glabra, recent photoassimilates and carbohydrate reserves in the wood of 49-11 were mobilized preferentially towards new shoot growth, whereas in A. glabra seedlings, they were preferentially mobilized towards plant organs other than new vegetative shoots. A more detailed examination of the eects of cyclic and continuous ooding on assimilate partitioning of Annona species is warranted. Growth rates of non-ooded A. squamosa seedlings were comparable to those reported for this species by Higuchi et al. (1998), who recorded growth of trees at 30\25 mC (day\night temperatures) in a naturally sun-lit glasshouse. The fact that trees of 4-5 \A. reticulata and Gefner \A. squamosa did not regain normal growth rates even 4 weeks after suspending ooding (expt 2) probably indicates that severe damage had been caused to both phloem and xylem tissues by that time. Indeed, all trees of these scion\rootstock combinations died within 2 months after nal growth measurements were made. Results indicate that the role of the root system as supplier of nutrient ions, metabolites, and phytohormones to the plant canopy (Jackson and Kowalewska, 1983) was irreversibly impaired in these Annona trees as a result of 30 d of continuous ooding. Similar responses to ooding have been documented for other susceptible tree species, including perennial and deciduous fruit trees (Coutts, 1981 ; Andersen et al., 1984 ; Schaer et al., 1992). In contrast, photosynthetic rates and subsequent growth of the tropical fruit species, A errhoa carambola L., recovered after relief of a 14 d ooding period (Razi and Mohd, 1996). Experiments 2 and 3 corroborated the ood tolerance of

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

tissues (mainly cortex) of submerged roots of many herbaceous woody plants (Coutts and Armstrong, 1976 ; Laan et al., 1989). The secondary tissues (cork and xylem) of a few mangroves and swamp-dwelling trees have aerenchyma (Fahn, 1990). In these rare examples, pneumatophores (which are modied roots) have a secondary xylem composed of thin-walled bres and much parenchyma. However, in some species, intercellular air spaces in the wood were no larger than usual (Fahn, 1990). Even in the most swollen stems with the least dense wood (ooded 49-11\A. glabra) there were no discernible air spaces between cells of xylem bres, xylem parenchyma, or xylem ray parenchyma. The bark also consisted of densely packed cells, including a compact green cortex and thin peripheral cork layer. Long-term ooding increased xylem cell size rather than cell number, and this produced thicker stems with less dense xylem. Flooding did not result in air spaces in pre-existing xylem near the pith, or in later xylem produced after ooding was imposed. The trunk base of intact A. glabra seedling trees and A. glabra rootstock ( 4911 \A. glabra) had a similar structure for ooded and non-ooded treatments. Therefore, survival and growth of ooded rootstock does not seem to involve production of new air canals, pathways, or intercellular air spaces in the wood or bark of ooded stems at the water line. Other morphological adaptations to ooding in A. glabra included outgrowth of new roots into oodwater and the development of hypertrophied lenticels on submerged roots. These structural responses would allow increased diusion of gases through the bark of the root. Development of hypertrophied lenticels as a result of ooding is a common response in ood-tolerant woody trees (Hook et al., 1970 ; Hook and Brown, 1973) including the tropical fruit species, Mangifera indica L. (Larson et al., 1991 a, b). The morphological adaptations of A. glabra, coupled with the relatively high A rates observed under ooded conditions, undoubtedly enable this Annona species to thrive in waterlogged environments for prolonged periods, as is the case in its natural habitat (Zotz et al., 1997). Adaptations to ooding in A. muricata included enhanced shoot production near the base of ooded stems !n (Nu 4 ez-Elisea et al., 1999). The decrease in wood density and cell wall lignication in ooded stems observed in this study has been reported for other woody plants (Schaer et al., 1992). The hydraulic architecture of stems in A. glabra was described by Zotz et al. (1997), although that study did not compare ooded s. non-ooded trees. This study has demonstrated large variation in oodtolerance among species of the genus Annona. A. glabra and A. muricata were highly ood-tolerant, while A. squamosa and A. reticulata were not tolerant to ooded conditions. A. muricata is reported to be adapted to areas with high rainfall (up to 4000 mm per year), but not tolerant of poor soil drainage (Villachica, 1996). A. glabra rootstock allowed survival and growth of ood-intolerant scions under ooded conditions. This nding suggests that commercial Annona species grafted onto A. glabra rootstock may thrive in periodically-ooded wetlands. Stem thickening and production of hypertrophied lenticels appear to be key

779

adaptations of A. glabra to ooding. However, enhanced girth at the stem base did not appear to involve aerenchyma formation, a common adaptive response of many plants to ooding (Kozlowski, 1997). A C K N O W L E D G E M E N TS We thank Mr Gary Zill (Zill High Performance Plants, Boynton Beach, Florida) for providing the plant material used in the present study. We also thank Lilia Caldeira and Miriam C. Mart! nez for help with data collection and plant and glasshouse maintenance. Florida Agricultural Experiment Station Journal Series No. R-06928. L I T E R A T U R E C I T ED
Andersen PC, Lombard PB, Westwood MN. 1984. Leaf conductance, growth and survival of willow and deciduous fruit tree species under ooded soil conditions. Journal of the American Society for Horticultural Science 109 : 132138. Campbell CW, Phillips RL. 1994. The Atemoya. University of FloridaFlorida Cooperative Extension Service Fact Sheet HS-64. Coutts MP. 1981. Eects of waterlogging on water relations of actively growing and dormant Sitka spruce seedlings. Annals of Botany 47 : 747753. Coutts MP, Armstrong W. 1976. Role of oxygen transport in the tolerance of trees to waterlogging. In : Cannell MGR, Last FT, eds. Tree physiology and yield impro ement. New York : Academic Press, 361385. Crane JH, Davies FS. 1988. Flooding duration and seasonal eects on growth and development of young rabbiteye blueberry plants. Journal of the American Society for Horticultural Science 113 : 180184. Drew MC. 1990. Sensing soil oxygen. Plant, Cell and En ironment 13 : 681693. Fahn A. 1990. Plant anatomy. 4th edn. Oxford : Pergamon Press. George AP, Nissen RJ. 1991. Eects of cincturing, defoliation, and summer pruning on vegetative growth and owering of custard apple (Annona cherimolaiAnnona squamosa) in subtropical Queensland. Australian Journal for Experimental Agriculture 27 : 915921. Higuchi H, Utsunomiya N, Sakuratani T. 1998. Eects of temperature on growth, dry matter production and CO assimilation in # cherimoya (A. cherimola Mill.) and sugar apple (A. squamosa L.) seedlings. Scientia Horticulturae 73 : 8997. Hook DD, Brown CL. 1973. Root adaptations and relative ood tolerance of ve hardwood species. Forest Science 19 : 225229. Hook DD, Brown CL, Kormanik PP. 1970. Lenticel and water root development of swamp tupelo under various ooding conditions. Botanical Gazette 131 : 217224. Jackson MB, Kowalewska AKB. 1983. Positive and negative messages from roots induce foliar dessication and stomatal closure in ooded pea plants. Journal of Experimental Botany 34 : 493506. Kennard WC, Winters HF. 1960. Some fruits and nuts for the tropics. Agricultural Research Service, United States Department of Agriculture, Miscellaneous Publication No. 801. Kozlowski TT. 1997. Responses of woody plants to ooding and salinity. Tree Physiology Monograph No. 1. Victoria : Heron Publishing. Laan P, Berrevoets MJ, Lythe S, Armstrong W, Blom CWPM. 1989. Root morphology and aerenchyma formation as indicators of the ood-tolerance of Rumex species. Journal of Ecology 77 : 693703. Larson KD, Davies FS, Schaer B. 1991 a. Floodwater temperature and stem lenticel hypertrophy in Mangifera indica (Anacardiaceae). American Journal of Botany 78 : 13971403. Larson KD, Schaer B, Davies FS. 1991 b. Flooding, leaf gas exchange and growth of mango trees in containers. Journal of the American Society for Horticultural Science 116 : 156160. Morton JF. 1987. Fruits of warm climates. Miami : J. F. Morton Publishers.

780

NuT n g ez-Elisea et al.Flooding Responses in Annona Species

Rowe RN, Beardsell DV. 1973. Waterlogging of fruit trees. Horticultural Abstracts 43 : 533548. Sanewski G. 1991. Custard apples, culti ation and protection. Brisbane : Queensland Department of Primary Industries. SAS Institute. 1996. SAS\STAT users guide. Cary, North Carolina, USA : SAS Institute. Schaer B. 1998. Flooding responses and water-use eciency of subtropical and tropical fruit trees in an environmentally-sensitive wetland. Annals of Botany 81 : 475481. Schaer B, Andersen PC, Ploetz RC. 1992. Responses of fruit crops to ooding. Horticultural Re iews 13 : 257313. Sena Gomes AR, Kozlowski, TT. 1980. Responses of Pinus halepensis seedlings to ooding. Canadian Journal for Forestry Research 10 : 308311. Villachica H. 1996. Frutales y hortalizas promisorios de la Amazonia. ! n Amazo ! nica, Secretar! a Pro-Tempore : Tratado de Cooperacio Food and Agriculture Organization. Zotz G, Tyree MT, Patin 4 o S. 1997. Hydraulic architecture and water relations of a ood-tolerant tropical tree, Annona glabra. Tree Physiology 17 : 359365.

Nakasone HY, Paull RE. 1998. Tropical fruits. Crop production science in horticulture. Wallingford : CAB International. Noble CV, Drew RW, Slabaugh JD. 1996. Soil Sur ey of Dade County Area Florida, U.S. Washington, D.C. : Department of Agriculture NRCS. Noonan JC. 1953. Review of investigations on the annona species. Proceedings of the Florida State Horticultural Society 66 : 205210. Nu! n 4 ez-Elisea R, Schaer B, Crane JH, Colls AM. 1999. Impact of ooding on Annona species. Proceedings of the Florida State Horticultural Society 111 : 317319. Ponnamperuna FN. 1984. Eects of ooding on soils. In : Kozlowski TT, ed. Flooding and plant growth. New York : Academic Press, 1045. Popenoe W. 1920. Manual of tropical and subtropical fruits. London : Hafner Press, Macmillan Publishers. Razi IM, Mohd NK. 1996. Growth and physiological processes of young starfruit (A errhoa carambola L.) plants under soil ooding. Scientia Horticulturae 65 : 229238.