Cost of inoculation seedlings with Pisolithus tinctorius spores

Although the production of commercial products of vegetative Pt (Pisolithus tinctorius (Pers.) Coker & Couch) inoculums has ceased in North America due to a lack of demand by consumers, the number of products that contain Pt spores has increased. The quality, quantity and price of these products vary considerably. The cost of inoculating 1,000 tree seedlings with Pt basidiospores can vary from $0.45 to more than $30. The cost of treating with Pt spores is lowest when seedlings are inoculated in a container nursery using rates that are less than 0.4 mg per seedling. However, with some products the cost to treat 1,000 bareroot seedlings is greater than $500 when spores are applied in the planting hole. Three decades ago, 1 g of Pt spores could be purchased for $0.13 and now the price of 1 g can exceed $14. Although many research papers provide data on the biological response to inoculating seedlings with spores, few document the cost of inoculation. Commercial products that are marketed toward homeowners containing both ectomycorrhizal and endomycorrhizal spores are more expensive than products that contain only ectomycorrhizal spores. In situations where survival and growth of seedlings are not increased, the benefit/cost ratio will typically be less than one.


Introduction
At some nurseries Pt (Pisolithus tinctorius (Pers.) Coker & Couch) mycorrhiza will form naturally on pine roots (figure 1). In some situations, adding this fungal symbiont to the growing media will increase the growth of pine seedlings in bareroot and container nurseries. In the past, some nursery managers collected fruiting bodies and then applied Pt spores to seedbeds in hopes of increasing the amount of mycorrhiza. For example, more than three decades ago nursery managers in South Africa (Strubbings 1958;Young 1981) and Oklahoma (Mexal 1980) applied Pt spores to nursery soil.
Pt research prior to 1970 was limited (Marx and Bryan 1969;Zak and Bryan 1963) but since that time, interest in conducting research with this symbiont has increased exponentially (figure 2). Positive results from nursery trials during the 20 th century likely explain why the number of commercial products using Pt spores has increased over time. Although several nursery manuals mention the use of mycorrhizal products, most nursery managers today do not purchase or apply commercial products that contain Pt spores. This raises the question: why has the number of commercial Pt products increased, yet the use in forest tree nurseries decreased? This paper summarizes research regarding applying Pt spores in tree nurseries and provides some reasons why nursery managers are reluctant to purchase and use commercially available products. Figure 1. The amount of Pisolithus tinctorius on short roots in October can affect both the color and mass of Pinus taeda seedlings that had roots cut in August (South et al. 1989). Green and yellow seedlings had Pisolithus tinctorius on about 67 percent and 1 percent of the seedlings, respectively. The percentage of short roots that were mycorrhizal were 51 percent and 31 percent for green and yellow seedlings, respectively. Root of yellow seedlings were dominated by Telephora terrestris mycorrhiza and green seedlings had about 20 percent more dry mass than yellow seedlings. The locations of Pisolithus tinctorius basidiocarps are indicated with white flags. (Photo by David South, 1983).
Figure 2. The trend in research papers that include the words Pisolithus tinctorius. Black bars represent papers that also include the word "spore".

Spores in bareroot seedbeds
When ectomycorrhizal inocula are absent from seedbeds and surrounding areas (Hatch 1936;McComb and Griffith 1946;Wakeley 1954), adding Pt spores to seedbeds at time of sowing can increase seedling growth. At one bareroot nursery with low levels of native mycorrhiza, applying Pt spores at time of sowing increased seedling mass by 12 percent to 26 percent (Marx et al. 1979). In contrast, when seedlings have ectomycorrhiza on 35 to 49 percent of the short roots, then adding Pt spores might increase the amount by 8 percentage points (table 1), but this might have no effect on seedling mass (Marx et al. 1989b). Thus, a growth response may be achieved when spore and mycelia counts are low in the rhizosphere, but benefits may not occur at nurseries with naturally occurring ectomycorrhiza (Cram et al. 1999;Castellano and Trappe 1991). It is not uncommon for harvested seedlings to naturally have more than 30 percent ectomycorrhiza (i.e. percent of short roots that have a Hartig net).
Inoculations with Pt spores provide little benefit at nurseries that have adequate soil inocula ) and high soil fertility (Cram et al. 1999;Cram and Dumroese 2012;Cordell et al. 1974) or when Pt spores have been stored improperly Trappe 1983a, Marx et al. 1986). Although some report that fertilization will reduce the percentage of ectomycorrhizal short roots, nitrogen applications totaling 300 kg/ha/yr or more do not inhibit Pt ectomycorrhiza of loblolly pine (Pinus taeda L.) (Marx 1990;Marx et al. 1979). In addition, soil phosphorus levels up to 150 ppm do not prevent the formation of ectomycorrhizae in pine seedbeds (Marx et al. 1989a; figure 3). In contrast, certain fungicides will inhibit the formation of Pt ectomycorrhiza. Table 1. A list of trials with Pisolithus tinctorius spores in bareroot pine seedbeds that were not treated with the fungicide triadimefon. Testing of spore inocula in bareroot seedbeds in the southern United States declined after these tests were published, in part, because the use of triadimefon fungicide increased. Within a row, an asterisk (*) indicates applying spores increased the level of ectomycorrhiza (α =0.05).
Figure 3. Operational data (control plots) from bareroot conifer seedlings growing in different nursery soils (n=41) indicate an unpredictable relationship between soil phosphorus at sowing and frequency of ectomycorrhizal short roots at time of harvest (adapted from Marx et al. 1984a). When soil phosphorus was less than 150 ppm (double acid extraction), the ectomycorrhizal short roots averaged 32 percent (n = 40). In various inoculation trials, the fungicide triadimefon was not used because it can inhibit the development of Pt ectomycorrhiza (Marx et al. 1986;Marx et al. 1989a, Rowan 1984. When certain other fungicides are used, the application of Pt spores may increase ectomycorrhizal short roots by as much as 10 percentage points. However, when triadimefon is applied to seedlings, the formation of Pt ectomycorrhiza is suppressed and other ectomycorrhiza short roots are reduced by 9 percentage points or more (Kelley 1987;Marx et al. 1986;Rowan 1984). Managers in the southern United States routinely apply triadimefon to control fusiform rust caused by Cronarium quercuum (Berk.) Miyabe ex Shirai F. sp. fusiforme Burdsall and Snow (Carey and Kelley 1993;South et al. 2016). This is because managers would rather produce seedlings with only 20 percent ectomycorrhizal short roots than to spend time culling diseased seedlings. In fact, sometimes outplanting survival is increased when seedlings are treated with triadimefon (Rowan 1984). Observations suggest that use of triadimefon has resulted in a decline in the production of Pt basidiocarps in bareroot nurseries. In the past, the occurrence of Pt basidiocarps was common at some bareroot nurseries (South et al. 1989). Despite the negative effects on Pt ectomycorrhiza, the benefits of rust fungicides to pine seedlings outweigh such concerns (Cram and Dumroese 2012;Rowan 1984).

Spores in containers
The growth response from applying Pt spores to container media depends on the level of native basidiospores in the surrounding environment. When there are no airborne ectomycorrhizal spores, untreated pine seedlings will likely be nonmycorrhizal and stunted, especially when the growing media has no inoculum. However, to produce conifer seedlings without ectomycorrhiza requires the potting media to be free of live fungal spores. For this reason, mycorrhizal researchers may sterilize media using fumigation (Mitchell et al. 1984;Ruehle 1980) or heat (Ambriz et al. 2010;Báez-Pérez et al. 2017;Lu et al. 1998;Molina, R. 1979). In addition, some researchers use special procedures to keep airborne spores away from germinating seedlings (Hatch 1936;Marx and Bryan 1969;Stottlemyer et al. 2008). For example, Marx (1976) steamed media three times and then kept the containers in an electronically air-filtered plant growth room. As a result, non-inoculated seedlings were stunted, had no mycorrhiza and were about 36 percent smaller five months after sowing. In greenhouses located in regions with a lack of spore contamination, seedlings growing in twice sterilized sand may be 26 percent smaller than seedlings treated with Pisolithus spores (Lu et al. 1998).
At many locations, airborne basidiospores will naturally inoculate container media and seedlings will not be stunted (Cram and Dumroese 2012). As a result, artificially inoculating containers with Pt spores may result in no increase in height growth (Trofymow and van den Driessche 1991;Whitesell et al. 1992; table 2). For example, in one test non-inoculated seedlings had ectomycorrhiza on 21 percent of the short roots (at age 16 weeks) and seedling mass (3.3 mg) was the same as for seedlings treated with Pt spores (Marx et al. 1984b). Likewise, container-grown oak seedlings (in unsterilized peat:vermiculite media) were not affected by adding Pt spores (Boling et al. 2006). Table 2. Examples of the effect of adding spores to height of container-grown pine seedlings in Spain and in the United States. Pinus taeda and Pinus ponderosa seedlings were grown in the USA while other species were grown in Spain. In Spain, adding Pt spores to container media may increase seedling height (>2 cm) in greenhouses about 30 percent of the time while in the USA an increase in height growth is rare.

Spores per seedling
The cost of using commercial Pt products depends on the number of spores applied per seedling. Most researchers apply high rates of spores that are not economical for operational nurseries. For example, some researchers applied more than 50 mg per seedling (Ambriz et al. 2010;Beckjord et al. 1986;González-Ochoa et al. 2003;Jorgensen 2014;Marx and Bryan 1975). Since it might cost $30 to apply just 1 g of Pt spores (figure 4), applying 50 mg of a commercial product would cost $1.50 per seedling. To reduce the cost, the rate per seedling was lowered, in some cases, by 98 percent. The rate selected for encapsulated seed was 1.1 million spores per seedling (Marx et al. 1984b) and a spore pellet contained approximately 3 million spores (Marx and Bell 1985;Walker and McLaughlin 1991). Although 27 research studies (out of 36 examined) used rates of at least 1 mg per seedling, most commercial products labels suggest applying lower rates.
While one nursery guide recommends 0.5 mg per seedling (Barnett and McGilvray 1997), nursery managers decrease costs by applying lower rates. In one trial, applying 0.15 mg of spores per seedling resulted in 67 percent of the pine seedlings with some Pt mycorrhiza (Preve et al. 1984) and 0.1 mg per seedling proved effective in two trials (figure 4). In 2017, one manager applied 0.03 mg per seedling to his entire seedling crop (Free 2017).
Commercial products vary widely in the amount of spores they contain. One product contains 30,000 Pt spores per kg and the label lists 99.9 percent inert ingredients. When this product is used as a side dressing, the recommended rate averages 7.5 to 15 spores m -2 . At 250 seedlings m -2 , this is equivalent to as few as one spore per 33 seedlings. This rate would not cause a measurable growth benefit. Figure 4. The cost of inoculation with Pisolithus tinctorius spores is directly related to the amount of spores applied per container (bars). When 1 g costs $30, then applying 1 mg of spores will cost $0.03 per seedling. The upper, middle and lower lines for percent ectomycorrhizal short roots are from Pera et al. (1994), Rincon et al. (2001) and Marx (1976), respectively.

Spores in planting hole
Adding Pt spores in the planting hole is both ineffective and costly. It is costly because often more spores are applied in comparison to typical rates used by nursery managers (Repáč 2011). It is ineffective because successful invasion by native mycorrhizal mycelia (colonizing seedling roots) can be a major problem in obtaining a desired response (Garcia- Barreda et al. 2015). For example, applying Pt spores to planting holes in non-fumigated forest soils failed to increase growth (Beckjord and McIntosh 1984;Bryson 1980;Davis and Jacobs 2004;Pilz and Znerold 1986;Wood 1985). At one location, applying too many spores to roots at time of transplanting reduced the survival of conifers (Alverez and Trappe 1983b). Thus far, research trials have not detected a significant growth benefit from adding Pt spores either in the planting hole (Beckjord et al. 1984) or when potting nursery stock (Weissenhorn 2002). Simply adding soil to the planting hole (Amaranthus and Perry 1987;Colinas et al. 1994;Helm and Carling 1993;Querejeta et al. 1998;Roldán et al. 1996) may have a higher probability of a positive seedling growth response.
Several Pt products that are marketed as a benefit to transplanting also include fertilizers. When a fertilizer tablet is applied in the planting hole, it will sometimes increase seedling growth (Hatchell and Marx 1987;Walker 2002). When mycorrhizal spores are added to a fertilizer tablet, the cost per ha might increase by 300 percent or more. If extra growth does occur, it is usually not known if the addition of spores played any role in increasing height growth.
Several products (targeted for use in planting holes) include additional compounds such as kelp, seaweed, humic acids, bone meal and vitamins. One information sheet says "if you think one, or two, aren't important, forget them and focus on the rest!" In fact, some do focus on the positive effects from using hydroscopic gels (Crous 2017;Sarvaš et al. 2007;Starkey et al. 2014) or applying more chemical fertilizers in the nursery (Davis et al. 2011;Marx 1990;South and Donald 2002). In fact, the British Standards Institute (Anonymous 2014) says "there is little literature to support the value of adding commercial mycorrhizal cocktails to the backfill soil used for young tree planting." Their recommendation agrees with Wilde (1944) who said "the question of mycorrhiza in the routine practice of silviculture has been unduly exaggerated".

Cost of inocula in 1990
The use of ectomycorrhizal inoculants in tree nurseries increased during the second half of the 20 th Century and at least ten companies were producing ectomycorrhizal inocula (Rossi et al. 2007). In North America, about five out of 78 container nurseries had an active inoculation program (Castellano and Molina 1990). The company Mycorr Tech, Inc. (Worthington, PA) sold vegetative inoculums at a price of $10 per liter and more than 6 million seedlings were treated using this product (Cordell et al. 1989). When mixed in the seedbed at a rate of 1.08 liter m -2 (and with 250 seedlings m -2 ), this added about 4.3 cents to the cost of producing a seedling (Marx et al. 1984). This treatment increased the number of Pt short roots by about 4 percent (figure 5). Without any Pt inoculation, pine seedlings typically have about 30 percent mycorrhizal short roots, primarily from species other than Pisolithus tinctorius (Marx et al. 1984a). Figure 5. A commercial vegetative inoculum (Abbot Laboratories -1.08 l m -2 ) of Pisolithus tinctorius (Pt) was applied to 37 pine seedbeds (Marx et al. 1984a) at a cost of about 4.3 cents per plantable seedling. Seedlings lifted from this treatment had, on average, 3.5 percent of short roots infected with Pt ( □ ) while 24 percent were infected with other species ( ■ ). There was no strong relationship between soil pH and frequency of mycorrhizal roots. On average, non-inoculated pine seedlings (not shown) had an average of 31 percent mycorrhizal short roots (range = 9 percent to 61 percent).
For some oak species (Pope 1988), applying 2.2 liters m -2 might cost 28.8 cents per seedling, doubling the price of seedlings. As a result, methods were developed to apply lower rates. The cost for pine seedlings was reduced by 63 percent when the

Other
Pt rate was lowered to 0.4 liter m -2 (Marx and Cordell 1988). When applying vegetative inocula it might take 6 weeks to form mycorrhizae compared to 15 weeks when applying Pt spores (Marx et al. 1979).
Applying spores to either bareroot seedbeds or to growth media (in containers) was a less expensive method of inoculation (Castellano and Molina 1990;Lu et al. 1998). To make spore application easier, methods were developed to encapsulate seed with spores (Marx et al. 1984b) and to make spore pellets. Sporeencapsulated seeds were marketed by SouthPine Inc. (Birmingham, AL) and by International Forest Seed Co. (Odenville, AL). In comparison to the cost of vegetative inoculum, the cost of using encapsulated seeds was up to 78 percent less expensive (table 3; Marx and Cordell 1988). In one trial, adding spores to encapsulated seed reduced seedling size because seedling emergence was reduced by about 3 weeks (Marx et al. 1984b). In 1995; a MycorTree™ Pt spore spray kit was marketed by Plant Health Care Inc. (Pittsburg, PA) and contained approximately 250,000 spores per mg of product (Martin et al. 2003).
Pt spores were not commercially available in 1978, but a decade later doublesifted spores could be purchased for $132 kg -1 (table 3). To supply companies with enough Pt spores, some mushroom collectors were paid $33 kg -1 for Pt sporocarps (figure 6). When collected at the right time, one large sporocarp might weight 200 g and would be worth more than $6 when harvested and $26 after spores were sifted. Estimates of the mass of Pt spores vary from 1.1 to 1.2 billion (10 9 ) spores per g Trappe 1983a, Marx 1976).

Cost of inocula in 2017
Significant changes have occurred in the cost and availability of Pt spores over the past three decades. In 1993 ectomycorrhizal spores could be purchased from Forest Mycorrhizal Application (Grants Pass, OR) at a cost equivalent to 1.5 cents per seedling (Landis 1993) at a price of about $0.20 per gram. Today, the price has increased and a gram of Pt spores is sometimes sold in products for $15 or more. A price offered on the internet was $40 per gram. As a comparison, a bareroot loblolly pine seedling in 1978 could be purchased for 1.2 cents and now one seedling will cost about 6 cents. There are currently numerous commercial products that include Pt spores and a few organizations provide "pure-culture" inocula (Wilkinson and Janos 2014). There is a wide range in the value of these products as one gram of product may contain more than 5 million spores while others contain less than 60 spores per gram (Wiseman et al. 2009). Most of these products are marketed for use in landscape areas, horticultural nurseries and homeowners. For this reason, most of the products that contain Pt spores also contain more expensive endomycorrhizal spores (table 4). This increases the range of tree species that may be added to the label. However, a few mycorrhizal products that are marketed as "general purpose" contain no ectomycorrhizal spores so, as with most products, it is important to read and understand the label.
The commercial price of endomycorrhizal spores is greater than Pt spores. This is because it is much easier to harvest a billion Pt spores, and because Pt spores are not as effective on a per spore basis (Báez-Pérez et al. 2017). In some cases, a penny will purchase either 58 endomycorrhizal spores (Cram and Fraedrich 2015) or 540 Pt spores. Therefore, adding endomycorrhizal spores to a commercial product increases the overall cost without providing any additional benefit to pine seedlings. In one example, 83 percent of the price per kg of a product was due to endomycorrhizal spores. A kg of a product that contains 132,000 endomycorrhizal spores might cost $13.55 while adding 110 million ectomycorrhizal spores might increase the price by $2.84. Products that contain only endomycorrhizal spores are occasionally used in nurseries at a cost of almost $20,000 per ha with little or no beneficial results to seedlings (Cram and Fraedrich 2015;Kűlling 2008;Wiseman et al. 2009).
In 2013-2014, five container nurseries were applying Pt spores to seedlings on an operational base. These nurseries were located in Alabama (Westervelt), Florida (IFCO), Georgia (IFCO), Louisiana (IFCO) and Nebraska (USFS Bessey Nursery). The cost of applying commercial products at these nurseries ranged from $1 to $3.45 per thousand. One liter of product was used to treat 130,000 to 250,000 seedlings. Since triadimefon was used at the southern nurseries, spores were applied (as a drench about the end of August) after the fungicide applications ceased. This fungicide was not used at the Bessey Nursery and, therefore, spores were applied at time of sowing. Managers indicate the treatments increased the production of Pt ectomycorrhiza but there was no noticeable effect on the overall amount of ectomycorrhiza. The additional cost did not produce a documented effect on seedling sales (especially in years when demand for seedlings was greater than the supply). As a result, only two nurseries applied Pt spores to 9 million container-grown pine seedlings in 2017. Table 4. A partial list of commercial products that contain spores of Pisolithus tinctorius (Pt). The cost per thousand seedlings is determined using the cost per billion Pt spores and an arbitrary rate of Pt spores. Some products are applied in the nursery (N), the gels are applied to roots (R) and some products are applied in the planting hole (H). Many products contain more than one species (#) of ectomycorrhizal spores (ECT) and several species of endomycorrhizal spores (END) and some products also include fertilizers (FERT). In some products, Pt spores represent 45 percent of all ectomycorrhizal spores.

Economic benefit
The economic benefit of commercial products "should be calculated prior to the decision to use mycorrhizal products" (Vosátka et al. 2008). Simply saying a particular treatment is "worthwhile" is insufficient. This is because some products may prove to be ineffective (Anonymous 2014;Cram and Fraedrick 2015;DeMuro et al. 1990;Maltz and Treseder 2015;Marx et al. 1984a, Repáč 2011Repáč et al. 2014;Vosátka et al. 2008). Profits can't be made when products do not work (Dettweiler-Robinson et al. 2013). Even when a treatment increases growth (Holuša et al. 2009), the treatment must cost less than the potential economic gains (Marx et al. 2002).
The total cost of applying Pt spores is easy to determine by adding the cost of application to the cost of materials. Once the total cost is determined, then estimates can be made of how much of a gain in crop value must be achieved in order to "break even." When the price per g is $30, and 1 mg of spores is applied per seedling, then the cost per seedling is $0.03. In order for some bareroot nurseries to "break even," they would have to raise the price of pine seedlings by 50 percent without losing any customers.
Applying Pt spores in containers might increase early height growth of pine seedlings about a quarter of the time (table 2). However, most managers in the southern United States top-prune seedlings to keep seedlings from growing too tall. Without the use of Pt spores, the median height growth of container-grown pines is 32 cm (South et al. 2016). Therefore, increasing height by an additional 1 or 2 cm may not be economical. In contrast, increasing seedling production is economical at nurseries where the costs do not exceed revenue from additional seedling sales (table  5). In the past, Pt spores were sometimes applied as a marketing tool (Landis 1993) in hopes this would attract customers away from other nurseries (thereby increasing seedling sales). Some suggest the use of nursery fertilizers can be reduced by applying mycorrhizal inocula (Dixon et al. 1981;Marx and Artman 1978;Sousa et al. 2012;Väre 1990). However, nursery managers know that fertilization is a relatively inexpensive way to achieve the target seedling in a limited amount of time. Applying a commercial Pt product is a more expensive method of increasing growth (figure 7). In one example (Khasa et al. 2001), spending 5 cents per seedling on vegetative Pt inocula (Coredell et al. 1989) reduced fertilizer cost by less than 0.1 cent per seedling (Clements and Dominy 1990;South and Zwolinski 1996). Likewise, when using fertilizers, applying Pt spores to containers appears to be an unreliable method of increasing height growth (table 2). Managers prefer to apply fertilizer to produce 30 cm tall seedlings (3.5 mm in diameter) than to spend more money to inoculate with Pt and produce smaller seedlings (23 cm height; 2.5 mm diameter) by using one-fourth the amount of fertilizer (Ruehle and Wells 1984).
Most researchers who publish papers do not include either the cost of Pt application or the effect of the treatment on the number of plantable seedlings produced. Therefore, researchers may test rates of Pt spores that are far from economical. As a result, nursery managers may need to collect their own data to determine any economic benefit of using lower dosages of Pt spores. A sound calculation should include (1) cost of spores, (2) cost of applying the spores, (3) effect on seedling production, and (4), probability of achieving an increase in production. The hardest part to determine is the probability of achieving an increase in production since it requires multiple tests conducted over several years. For example, in 21 tests with loblolly pine over a four-year period, a commercial (vegetative) Pt product caused a statistically significant ( =0.05) increase in plantable seedlings in only one test (Marx et al. 1984a). Likewise, when applying Pt spores at three Eucalyptus nurseries over a two-year period (Brundrett et al. 2005), inoculation resulted in Pt ectomycorrhiza in 2 out of 8 attempts. Variable results such as these and the discrepancy found between laboratory and nursery studies help to explain why some managers do not inoculate seedlings with Pt spores (Araujo et al. 2018). Figure 7. Although the cost of applying commercial products containing Pisolithus tinctorius (at one million spores per seedling) could exceed $15 per thousand seedlings, the cost of applying chemical fertilizers can cost less than $0.40 per thousand seedlings. In trials with Pinus pinaster (Rincón et al. 2007) and Quercus rubra (Beckjord et al. 1985), fertilized seedlings were more than twice as large as seedlings treated with only Pt spores.

Effect of Pt spores on outplanting performance
There are two schools of thought regarding the use of Pt spores in pine nurseries. One school "A" believes the application of spores in commercial products will increase the survival potential on adverse sites, while those from school "B" say the benefit/cost ratio of applying Pt spores will likely be less than 1 for many outplanting sites. Although several studies show that use of vegetative inoculants can sometimes increase survival of loblolly pine (Trofymow and van den Driessche 1991), only a few commercial products have been tested. Treating pines with Pt spores (at time of sowing) increased survival in just one of 14 outplanting trials (figure 8) and in New Mexico, treating seedlings 6 weeks after sowing increased survival of one out of seven species (Harrington et al. 2001). Spores did not increase survival of containergrown longleaf pine (Pinus palustris Mill.) but height growth was increased by 19 cm (4 years after planting) (Cram et al. 1999).
Some say that 50 to 60 percent infection with Pt is needed in order for a Pt treatment to achieve a significant increase in survival and growth (Marx et al. 1984b, Ruehle 1980. These Pt infection rates (short roots) may be difficult to achieve using Pt spores, especially at nurseries that use Pt inhibiting fungicides (Marx 1987). Even when alternative fungicides are applied to Pt spore treated seedlings, the Pt index of bareroot loblolly pine seedlings may range from 33 percent to 46 percent (Marx et al. 1989b). Therefore, to obtain a Pt index of 50 percent may require the culling of some (otherwise plantable) seedlings (Cram et al. 1999).
When discussing seedling quality, mycorrhizae are often not mentioned (Duryea 1984;Grossnickle 2012;Grossnickle and South 2017;Lavender, 1984;Puttonen 1996;Ritchie 1984;South 1993;Wilson and Jacobs 2005). This is because pine and oak seedlings typically have ectomycorrhiza and, at most operational nurseries, the percentage of short roots with mycorrhiza is not a reliable predictor of field performance. In fact, well fertilized container-grown seedlings (without Pt mycorrhiza) have outperformed smaller seedlings with plenty of Pt ectomycorriza (Barnett 1983;Echols et al. 1990). This type of response likely explains why some researchers (DeMuro et al. 1990;Repac et al. 2011;South and Skinner 1998) and most silviculturists do not count the number mycorrhizal short roots on nursery stock. Figure 8. Applying Pisolithus tinctorius spores before or just after sowing in the nursery typically does not increase seedling survival in the field. Only one of the paired tests was associated with a significant increase in survival (i.e. 8 percent above the diagonal line). Data are from loblolly pine (Pinus taeda), slash pine (Pinus elliottii), Virginia pine (Pinus virginiana) , slash pine (Rowan 1984), loblolly pine (Leach and Gresham 1983) and longleaf pine (Pinus palustrius) (Cram et al 1999).
10 The decline effect "Many scientifically discovered effects published in the literature seem to diminish with time" (Schooler 2011). When studies are repeated, the magnitude of the treatment response may be less than expected, based on the initial published results. This phenomenon was discovered in research into parapsychology but the phenomenon also occurs in biology. For example, South (1998) reported on 30 toppruning studies installed from 1949 to 1979. As it turned out, the initial 1949 study reported a 55 percent increase in survival, while none of the subsequent studies reported an increase of 37 percent or more.
This phenomenon may also exist with Pt spore research. In places like India, South Africa, Iowa and Hawaii, the amount of airborne spores was initially low and adding spores to nursery soil and media was beneficial (McComb 1943;Thapar and Paliwal 1982). However, the area of exotic conifer plantations increased over time and since the atmosphere now contains more mycorrhizal spores, artificial inoculation may no longer be necessary (Whitesell et al. 1992). A higher level of airborne spores is one explanation for the "decline effect." In addition, a decline phenomenon may also exist in regions with naturally high levels of airborne spores. In 1973; applying Pt spores to pine seedbeds at the Edwards Nursery in North Carolina increased total ectomycorhiza by 18 percentage points but since that time, an increase that large has only been documented once (table 1). An alternative explanation for "decline effect" exists because rates used operationally in 2017 were much lower than the 7 mg per seedling rate used in 1973.

Final thoughts
Although the production of commercial products containing Pt spores has increased globally, their use in North American tree nurseries has declined. At some nurseries, the decline occurred because the cost was greater than the benefits. High quality, ectomycorrhizal seedlings can be produced at most nurseries that are located in regions with sufficient levels of airborne spores. Although some researchers recommended combining high spore rates with reduced rates of fertilizers and restricting use of certain fungicides, most managers find profits are greater when these suggestions are ignored.