! Inoculations: Plants were inoculated when roots about 10 cm long had developed in the nutrient solution below the rockwool plugs. Inoculum

3 -1prepared for each Pythium isolate comprised 5x10 zoospores mL half strength nutrient solution. For inoculation, each plant was lifted from the hydroponic unit, its root system was immersed in a zoospore suspension for 30 min, and the plant was returned to the unit.

! General observations

! Isolate P6: Percent of roots discolored increased rapidly after initial appearance of root tip necrosis. Discoloration can begin anywhere along the length of the root. Swelling and callus formation in primary roots sometimes preceded necrosis. Rapid necrosis largely precluded lateral root proliferation and affected plant systems failed to grow.

! Isolate P8: After initial necrosis of root tips, yellowing of the roots occurred, later turning greyish, and an abundance of short lateral roots developed.

! Isolate P11: Primary roots with tip necrosis (~ 2 mm of root tips) developed numerous short lateral roots. These developed root tip discoloration soon after emergence. Regrowth of roots from discolored tips was frequent. Roots often thickened over time, and entire root systems appear yellowish in color.

! Isolate P15: Tips of roots are dark brown and necrotic. Lateral roots often long and thick. Callus formation sometimes occur at the base of primary roots and can extend to the rest of the root.

! Isolate P17: Primary roots swollen; root tips necrotic to ~ 5 mm; proliferation of lateral roots.

! Isolate P23: Lateral roots often long and fine. Callus formation occurs in increasing severity over time.

! Pythium isolates: Isolates were obtained from discolored young roots of hydroponic sweet pepper, lettuce and cucumber in commercial greenhouses.

! Experiment: Effects were examined of the six Pythium isolates on each of the following variables:

! Types of symptoms! Percent incidence of brown root tips! Percent of total roots that were discolored! Distribution of Pythium in roots as determined on a selective medium! Intensity of colonization, density of oospores, and proportion of

tissues with granular cell contents in roots examined microscopically

The experiment was conducted three times (April 2000, February 2001, May 2001). In each repetition the treatments and noninoculated checks were replicated three times in an RCBD. The data from each experiment were analyzed using analysis of variance (ANOVA) with alpha = 0.05.

Roots of noninoculated control plants were almost entirely whitish (Fig. 1A). Symptoms observed in root systems inoculated with the various Pythium isolates were:

! Root tip discoloration (all isolates, Fig. 1B);! Progressive discoloration of roots (Isolates P6 and P11 only, Fig. 1B,

1C);! Stunting (isolates P6, P8, and P11, Fig. 1B);! Proliferation of lateral roots (all isolates, Fig. 1B, 1D);! Swelling of roots (isolates P6, P8, P11, and P17, Fig. 1B);! Callus formation on primary roots (all except P11)

Progressive root necrosis (Fig. 3): Progressive root discoloration (browning or yellowing) during the 14 days of study was observed only for isolates P6 and P11. Necrosis of roots inoculated with P6 was progressive in all experimental repetitions, but much greater in the May study than in February or April. Yellowing in roots inoculated with P11 was progressive only in the April study. In the studies of February and May, discoloration generally increased during the initial four days after roots were inoculated but not thereafter. Similarly, discoloration in roots inoculated with P8, P15, P17 and P23 increased chiefly during the initial 4 days after inoculation. Severity of discoloration generally was greater in the study conducted in May than in those of February or April.

Distribution of Pythium in roots: All isolates produced colonies from all 1-cm segments that were cut from distal 10 cm long portions of primary roots at 3 and 9 days after inoculation and incubated on the selective medium.

Microscopic observations: Incidental fungi, chiefly Fusarium spp., were present in roots of the noninoculated controls and roots inoculated with the isolates of Pythium. In the controls, the incidental fungi gave a hyphal index near 20 (Fig. 4). Granular contents were frequent in host cells near to the hyphae. The incidental fungi produced no macroscopic symptoms.

Hyphal indices for roots inoculated with the Pythium isolates were, in each instance, high for all sites of observation from 0.5 cm to 9.5 cm from the root tip. Indices for host cell granulation were moderately high for all isolates and the checks, but in controls were generally lower in the regions closest to the root tip.

Oospore density was low in all isolates in this study.

! Disease progress

Incidence of brown root tips and root discoloration (Fig. 2): For each isolate, patterns of increase of brown root tips differed among the three experimental repetitions. Incidence of tip necrosis during 14 days after inoculation was relatively low for all isolates when the experiment was conducted in the winter (February). Conditions in April were highly favorable tfor tip necrosis in roots inoculated with P15 and P17, and moderately favorable for P8 and P11, but P6 produced about the same incidence of tip necrosis in April as in February. Conditions in May strongly favored P6 and P11, and were moderately favorable for all other isolates. Factors contributing to shapes of the curves included tip necrosis produced by zoospores of the initial inoculum, production of new roots with symptomless tips, and tip necrosis produced by zoospores and hyphae of subsequent inoculum.

Table 2: Root symptoms of Pythium isolates Treatment RTN RSD SRS PLR RS CF Control P6 · · · · · · P8 · · · · · · P11 · · · · ·

P15 · · · P17 · · · · P23 · · · RTN = root tip necrosis, RSD = root system discoloration, SRS = stunted root systems, PLR = profuse lateral roots, RS = root swelling, and CF = callus formation.

Table 1: Pythium isolates from hydroponics in Ontario Isolate # Species Host P6 Pythium aphanidermatum* Cucumber P8 Pythium dissotocum** Sweet pepper P11 Pythium dissotocum** Sweet pepper P15 Pythium dissotocum** Sweet pepper P17 Pythium dissotocum** Sweet pepper P23 Pythium dissotocum*** Lettuce

* From W.R. Jarvis ** Identified based on nucleotide sequencing of regions within the 5.8s ribosomal RNA gene, internal transcribed spacers 1 and 2 (IST1 and IST2), morphological and biometric features. *** From R. Evans

Results

! Symptoms:

Root Symptoms of Hydroponic Pepper Inoculated with Pythium Isolates1 2T.N. OWEN-GOING, J.C. Sutton, H. Yu and B. Grodzinski

Department of Environmental Biology and Plant Agriculture, University of Guelph, Guelph, Ontario, N1G2W3, CANADA

1 1

1 2

IntroductionRoot rot is a serious threat to productivity of sweet pepper (Capsicum annuum L.) in greenhouse hydroponic systems in southwestern Ontario, Canada. The peppers are produced in troughs formed from black-on-white plastic sheeting, usually without a rooting matrix, or in rockwool. Plant nutrient solution is circulated over the roots in the troughs or through the rockwool. Economic losses from root rot are sometimes major, especially when severe disease terminates production of marketable fruit.

Preliminary observations indicated that Pythium spp. are associated with root rot in the hydroponic peppers, as was reported for several other kinds of hydroponic crops (Jarvis 1992), and for field grown peppers (Chellemi et al., 2000). An improved understanding of Pythium spp. in relation to roots and root disease in hydroponic peppers is needed as a basis for developing biological and other methods to control root rot. Therefore Pythium spp. from hydroponic crops were examined for ability to colonize and cause symptoms in pepper plants grown in nutrient solution.

Methods! Pepper plants: Plants of sweet pepper cv. Merlin were grown in single-

plant hydroponic units. Each unit comprised a 1.9 L white plastic container inserted into a black plastic pot. The container lid, covered in black plastic, had a central hole (2 cm x 2 cm) to accommodate a pepper transplant in a rock-wool plug. Each container was kept filled with nutrient solution (pH 5.8,

-1electrical conductivity 1.5 mS cm ), which was aerated continuously by bubbling compressed air. Plants were maintained in a climate-controlled greenhouse at 20-23C with supplementary lighting to compensate for changes in daylength.

Conclusions! The various Pythium isolates, including those of the same species (P. dissotocum), produce characteristic and often differing symptoms in roots of hydroponic pepper. Browning of root tips is common to all isolates but area and discoloration intensity of affected tips differs among isolates. Expansive discoloration of root systems is brown for the P. aphanidermatum isolate but yellow for isolates of P. dissotocum. Patterns of lateral root proliferation and root system stunting each differ among isolates.

! Swollen primary roots and associated callus cells are common symptoms in hydroponic pepper infected by Pythium isolates.

! Patterns of increase in incidence of brown root tips and of percent discolored roots exhibit differences related to Pythium isolate and seasonal conditions in the greenhouse.

! All of the Pythium isolates can rapidly colonize major portions of inoculated root systems in hydroponic pepper.

ReferencesChellemi, D.O., Mitchell, D.J. and Kannwischer-Mitchell M.E. 2000. Pythium spp. associated

with bell pepper production in Florida. Plant Disease 84(12):1271-1274.

Jarvis, W.R. 1992. Managing diseases in greenhouse crops. APS Press, St. Paul, Minn.

AcknowledgementsThis research was funded by the Natural Sciences and Engineering Research Council of Canada and the Centre for Research on Earth and Space Technology (CRESTech). Travel funds were provided by CRESTech and University of Guelph travel scholarships (Taffy Davison Memorial; Arthur G. Latornell Memorial). We thank Kathia Marie Hallal and Dr. Weizhong Liu for assistance.

Figure 2: Incidence of brown root tips and; Figure 3: Root discoloration over time.

Figure 4: Colonization by Pythium

Figure 1: Control and Pythium-inoculated roots.

A B C D