Methodology for certification of Quercus ilex seedlings inoculated with Tuber melanosporum for commercial application

FISCHER, Christine (1) & COLINAS, Carlos (1)

Dep. de Investigación Forestal de Valonsadero, Apdo. 175, E-42080 Soria, Spain.

(1) Present address: Universitat de Lleida. Dept. de Producció Vegetal i Ciència Forestal. Av. Alcalde Rovira Roure, 177. E-25198 LLEIDA, Spain.

Poster Presentation at the1st International Conference on Mycorrhizae. Berkeley, California, U.S.A. August 1996.

Introduction

The decrease in edible truffle (Tuber sp.) production over the last 50 years from naturally occurring sites in France, Italy and Spain has promoted a great interest in cultivation. Commercial inoculation methods have been employed in France and Italy since the 1970´s, with certification of seedling quality and mycorrhization managed by regulatory bodies of the appropriate country (Chevalier and Grente 1978; Bencivenga et al. 1987) with recent changes in the Italian certification method (Bencivenga, M., Donnini, D., Tanfulli, M., and Guiducci, M. 1995). In the past five years interest in truffle cultivation has increased in calcareous regions of Spain and a practical, economical method for certification of commercially inoculated seedlings of Spanish nursery production is needed.

Objectives

The objectives of our study were to 1) determine the minimum number of root apices necessary to evaluate a single plant in order to get a representative value for the proportion of Tuber–inoculated root tips (PT) and the proportion of contaminant to Tuber–inoculated root tips (PC); 2) determine the minimum number of plants necessary to evaluate a plant lot in order to get a representative value for the proportion of Tuber–inoculated root tips (PT) and the proportion of contaminant to Tuber–inoculated root tips (PC) in the roots of plants in the lot; 3) develop guidelines to certify or reject a given plant lot based on plant quality and acceptable levels of mycorrhization by T. melanosporum and by contaminants.

Here we present the final method developed in this study.

Method

Identification of Plant Lot

Certification begins with the documentation of homogeneity and guarantee of plant health status as set by national standards. Homogeneity of plant lot requires that all plants have the following conditions in common: seed source, inoculation source and method, date of inoculation, substrate, and nursery conditions.

Sampling of Plant Lot

From each lot 12 plants are selected. This number is based on the variability among lots (Fischer and Colinas, 1996). It is essential that this selection be completed by the certifying agency and that it be done completely randomly in order to avoid any type of bias and to satisfy the assumptions of the statistical tests. Plants should be transferred to the certifying laboratory in coolers and stored at 4º C. until examined, preferably within 2 weeks of sampling time.

Cleaning Procedures

Plants should be carefully removed from the container with the substrate and root system intact and placed in a cool water bath to moisten the entire root system. Individually, plants are placed in a shallow pan of water with gentle agitation until the majority of the substrate is loosened and can be removed without damaging root tips and avoiding direct pressure from a stream of faucet water. Substrate particles which remain attached to the roots can be further eliminated with tweezers at the binocular microscope.

Evaluation of Plant Quality

Prior to evaluation of levels of mycorrhization it is necessary to verify that plants meet criteria for plant quality (Appendix 1). Plants which do not meet these basic standards will not be further evaluated in the more meticulous process of root tip counting. The European Community standards require that 95% of a given lot pass plant quality controls (Peñuelas 1993). The analyst will record the results of this evaluation in the Plant Data Sheet (Appendix 2).

Preliminary Observation at the Binocular Microscope

The second step in the evaluation process is to examine the entire root system at the binocular microscope, observing the abundance and condition of fine roots and mycorrhizae, the general appearance of the mycorrhizae of T. melanosporum, and the presence of contaminants. At this stage, the analyst should examine any root tips of doubtful identification by making slides for examination of microscopic characteristics. Presence of mycorrhizae of other species of Tuber will result in rejection of the lot. Three other criteria must be met at this stage: 1) a minimum of 250 healthy fine roots; 2) at least 10% mycorrhization by T. melanosporum ; 3) no more than 50% root tips obviously colonized by contaminant fungi. The analyst will record the results of this evaluation in the Plant Data Sheet(Appendix 2).

Counting of Fine Root Tips

Plants which pass the first two evaluations will be evaluated for the proportion of T. melanosporum mycorrhizae (PT) and the proportion of contaminating mycorrhizae (PC). The procedure is as follows:

1) Remove the stem from roots and cut the root system in half longitudinally, randomly choosing one half to examine.

2) Carefully cut the roots of the chosen half in 2-3 cm segments and place them over a 1 cm x 1cm grid in a shallow dish of water.

3) Spread these segments evenly over the grid and begin the count using squares chosen randomly.

4) In each square all fine roots are counted by category: Nonmycorrhiza (N), T. melanosporum mycorrhiza (T); Contaminating mycorrhiza (C). Senescent tips are counted as nonmycorrhiza.

5) Continue counts in proceeding squares until 250 tips are categorized (Fischer & Colinas, 1996).

The analyst will record the results of this evaluation in the Plant Data Sheet (Appendix 2).

Determination of Proportions

Based on the 250 counts the following proportions are calculated:

Proportion of T. mel. (PT) = T/(N+C)

Proportion of Contaminants (PC) = C/T

The analyst will record the results of these calculations on the Plant Data Sheet.

Determination of Confidence Intervals for PT and PC in a Lot (Appendix 3)

After completing the evaluations and calculations for PT and PC for 5 plants the analyst will determine the 95% confidence intervals for the values of PT and PC for the lot. If the criteria are met, the lot can be certified without further sampling. If not, the analyst should continue sampling until either the lot meets the criteria or the 12th plant is reached. If the lot does not meet the criteria then, it cannot be certified.

Criteria for Certification

Certification of a lot requires completion of the following criteria:

a) All plants sampled have completed plant quality criteria.

b) All plants sampled have completed the requirements for the Preliminary Observation.

c) No single plant within the sample has a PT < 0.11 (10% colonization by T. melanosporum).

d) No single plant within the sample has a PC > 1.0 (50% colonization by contaminants).

e) The lower limit for the Confidence Interval for PT > 0.50 (33% colonization by T. melanosporum ).

f) The upper limit for the Confidence Interval for PC < 0.33. (Contaminants = no more than 25% of colonized root tips).

g) No single plant within the sample has been colonized by species of Tuber other than T. melanosporum.

If a lot does not meet criteria e, f, and g, the lot will be rejected.

If a lot meets criteria e, f and g, but 1 or 2 plants do not meet criteria a, b, c, or d, the lot may be submitted for a second evaluation with a much larger sampling number if the nursery so chooses.

If a lot meets criteria e, f and g, but 3 or more plants do not meet criteria a, b, c, or d, the lot will be rejected.

Second Evaluation of a Large Sample Size

If we observe a single plant which does not meet plant quality standards or colonization criteria within a sample of only 12 plants, it is impossible to guarantee that the lot has less than 5% plants with these characteristics. For this, a sample size of up to 90 plants (Steel and Torrie 1980) is required. This larger sample is recommended only if the PT and PC of the first sample are sufficiently acceptable.

The process for the Second Evaluation is similar to the first with regard to evaluation of each plant according to criteria for plant quality and the preliminary observation at the binocular scope. Counting of root tips is not required if the plants pass these 2 stages. The confidence interval will depend on the number of plants analyzed and the number of plants rejected, using a binomial confidence interval table and an upper limit of 5% for plants rejected. (Steel and Torrie 1980). It is suggested that the analyst obtain confidence intervals every 5 or 10 plants after the 23rd plant, which is the first in which the amplitude of CI could be less than the limit. In this way it will be possible to accept or reject a lot prior to evaluating the entire sample of 90 plants.

Discussion

Certification of plant lots for colonization by Tuber ectomycorrhizae is a destructive and laborious process and therefore it is important to sample the minimum number of plants necessary to statistically satisfy that the established criteria are met. The first 2 stages in this method allow a skilled observer to rapidly and systematically reject plants and lots that obviously will not meet final criteria, and reduce the time-consuming process of root tip counting. We suggest that if the plants in a given lot appear homogenous and have acceptable values for PT and PC that the analyst compute confidence intervals for the lot after examining the first 5 plants of the lot instead of completing 12 samples. This method is designed to statistically admit quality lots with a minimum number of root-tip counting. Homogenous lots, with acceptable levels of PT and PC and small variances can be certified with less than 12 samples. Using this method, certification costs for a lot of 1000 plants, are estimated to be approximately 3% of the retail value of the inoculated seedlings (Appendix 4). This cost can be reduced as the method is further developed.

The threshold levels we have chosen for PT and PC are based on our observations of nursery-inoculated plants and by Italian standards (Bencivenga pers. com.). We have chosen a PT of 0.50 or 33% of root tips to be colonized by T. melanosporum. The PC of 0.33 sets the limit for contaminants to be, at most, 20% less than T. melanosporum ectomycorrhizae, and in addition, limits contaminants to not more than 25% of colonized root tips.

It will be necessary to test these results in the field in order to justify these thresholds under different field conditions. Ideally a certification program should include a research program in order to identify contaminants which present a serious threat to displacement of the desired ectomycorrhiza (Bencivenga et al. 1995).

Literature Cited

Bencivenga, M., Di Massimo, G., Donnini, D. and Tanfulli, M. 1995. Micorrize inquinanti frequenti nelle piante tartufigene: Nota 1-Inquinanti in vivaio. Mic. Ital. 2: 167-178.

Bencivenga, M., Donnini, D., Tanfulli, M., and Guiducci, M. 1995. Tecnica di campionamento delle radici e degli radicali per valutazione delle piante micorrizate. Mic. Ital., 2: 35-47.

Bencivenga, M., Ferrara, A.M., Fontana, A., Granetti, B., Gregorio, G., Lo Bue, G., Palenzona, M., Rebaudengo, E., Tocci, A. and Zambonelli, A. 1987. Valutazione dello stato di micorrizacione in piantine tartufigene. Ministero dell'Agricoltura e delle Foreste, Italy, 1987.

Chevalier, G. and Grente, J. 1978. Application pratique de la symbiose ectomycorrhizienne: production à grande échelle de plants mycorrhizés par la truffe (Tuber melanosporum Vitt.). Mushroom Science, 10: 483-505.

Fischer, C. and Colinas, C. 1996. Puesta a punto de un método de control de planta de Quercus ilex inoculada con Tuber melanosporum. Informe final. Centro de Investigación Forestal de Valonsadero. Junta de Castilla y León. Soria. Spain.

Peñuelas Rubira, J. 1993. Calidad de la planta forestal para el plan de reforestación de tierras agricolas. Montes, 33: 84-97.

Steel, R. and J. Torrie. 1980. Principles and Procedures of Statistics. McGraw-Hill Publishing Company. New York, NY, EE.UU.

Appendix 1: Criteria for evaluating plant quality of containerized Quercus ilex seedlings

Appendix 2: Plant Data Sheet

Appendix 3: Determination of Confidence Intervals for PT and PC in a lot.

Appendix 4: Estimate of cost of certification for one Quercus ilex seedling inoculated with Tuber melanosporum mycorrhizae