Protection of Highbush Blueberry Plants against Phytophthora cinnamomi Using Serendipita indica

: A greenhouse experiment was carried out on the use of Serendipita indica , an endomycorrhizal-like fungus, to increase the resistance of three highbush blueberry cultivars Chandler, Darrow and Spartan to the pathogen Phytophthora cinnamomi . The cultivars di ﬀ ered in the acceptance of root colonisation by S. indica and in susceptibility to P. cinnamomi . The lowest percentage (%) of colonised roots was found in the cultivar Spartan. The frequency of symptomatic plants (stunted growth, dark spots on stems, small, yellowing, and dropping leaves) among plants inoculated only with P. cinnamomi was 59, 83, and 100%, for ‘Chandler’, ‘Darrow’, and ‘Spartan’, respectively. In the treatment where both pathogen and endophyte were inoculated, the frequency in ‘Darrow’ and ‘Spartan’ dropped to 28 and 90%, respectively. Of non-protected ‘Spartan’ plants, 66% died, whereas only 14% of those protected with S. indica died. Colonisation by S. indica reduced the number of dead plants of ‘Chandler’ from 3 to 1 among those protected. In ‘Darrow’ only one plant died of the non-protected and none of the protected ones. S. indica increased the growth of non-infected plants of ‘Darrow’ and ‘Spartan’ by 11%. An increase in the growth of plants inoculated with S. indica and infected with P. cinnamomi after 21 days went from 74% to 182% over the plants only infected with the pathogen.


Introduction
Highbush blueberry (Vaccinium corymbosum) fruit is valued for its health-promoting potential. It is a rich source of anthocyanins, mineral salts, and vitamins [1,2]. The fruits of highbush blueberry contain on average about 200 mg/100 g Fresh Weight of anthocyanins [2,3]. Several cultivars are bred for various soil and climate requirements, enabling their cultivation in several climatic conditions in numerous countries [4]. About 76% of the world's highbush blueberry fruit is produced in the United States, Chile, and Canada in 2016 [5]. World production amounted to 861.55 metric tons in 2016, and the increase in production was 16.3% compared to 2014 [5]. Highbush blueberry is in the spotlight among European, South American, and Asian growers. This fruit is mostly consumed as fresh fruit, but processed products (frozen, jelly, saft) are also available on the market. The cultivation of highbush blueberry is the fastest growing direction of fruit production in Poland [6] where the interest in cultivation of highbush blueberry is growing due to its economic attractiveness and the fruits are sold at home and exported, mostly to the European Union. Poland produces approximately 21 thousand tons of these fruits, which ranks it a second in European (Statistics Poland 2017) and the seventh in global production of highbush blueberries [5].
Plants of the Ericaceae family are known for their high sensitivity to pathogens of the genus Phytophthora, including P. ramorum [7] and P. cinnamomi [8]. The method of plant production, on an industrial scale in nurseries and cropping fields, the need for frequent irrigation, and transfer of 3 of 11 substrate, P. cinnamomi was incubated in the dark for 7 days on an oatmeal substrate (5 g oatmeal and 15 mL distilled water per 9 cm diameter Petri plate, autoclaved twice for 30 min with an interval of 24 hours), homogenised, and added to the peat in a proportion of one Petri dish per 1 litre of peat. Then, after 2-3 weeks at 23 • C, 0.2 kg of the infected substrate was collected and the presence of P. cinnamomi zoospores was determined according to the procedure described by Themann [26], using rhododendron leaves as baits. Briefly, infected substrate was flooded with distilled water and leaves of rhododendron 'Nova Zembla' were placed on the surface of the water and incubated at 23 • C in the dark for 4-5 days. After this time, leaves were taken out, washed under tap next distilled water and dabbed dry. On each leaf blade number of necrotic spots were counted. In sterile laminar bench chosen leaves were sterilised over a burner flame and small fragments of tissue were cut from individual necrotic spots and placed on PDA medium (Potato Dextrose Agar, Merck KGaA, Darmstadt, Germany) in 90-mm Petri dishes for fungus identification, which were made as described below (see Section 2.6). After 7 days, 1 litre of infected substrate was mixed with 10 litres of pure peat substrate.

Inoculation of Highbush Blueberry Plants with S. indica and P. cinnamomi
For the experiment, blueberries were planted into 500 mL plastic pots with a peat substrate dedicated to Ericaceae, pH 3.5-4.0 (Alonet, Skierniewice, Poland). There were four combinations, with 29 plants each, as follows: 1-pure peat substrate, 2-substrate inoculated with S. indica, 3-substrate inoculated with P. cinnamomi, 4-250 mL of substrate inoculated with S. indica and after 21 days, supplementation with 250 mL of the substrate infected with P. cinnamomi. After planting, the substrate was carefully watered. The pots of single combination were placed in separate plastic boxes with a solid bottom lined with an underlying mat. During the winter, plants were maintained in a non-heated greenhouse. They were fertilised twice per year in April and May with Hydrovit (Hydrokomplet, Nowa Wieś, Poland) at a concentration of 1.25%. The experiment was terminated in October 2019. Observations of the symptoms of the disease, such as wilting leaves, leaf discoloration and fineness, and stem tip and shoot necrosis, were initially made on a weekly basis, then every month for 24 months.

Assessment of Colonisation of Highbush Blueberry Roots by S. indica
The evaluation of root colonisation was carried out for combinations 2 and 4, after 9 months (5 plants were randomly chosen) and at the end of the experiment (10 plants). From each plant, 1.5 cm root fragment was collected from 10 roots. They were thoroughly washed with distilled water to remove the peat substrate. Root surfaces were sterilised in a solution containing 4 mL laurylsarcosin 20% + 32 mL Ace whitening preparation (about 5% of sodium hypochlorite) and 64 mL of sterile water, as recommended by Johnson [23]. The presence or absence of S. indica chlamydospores in the 10 fields of view in each of the extracted root fragments was assessed, what means observation on 50 and 100 fragments (500 and 1000 fields of view). Root preparations were stained with cotton blue [27] or analysed without staining under a light microscope Eclipse 80i (Nikon, Tokyo, Japan) at a magnification of ×125.

Re-Isolation of P. cinnamomi from Infected Plants and Substrates
Fragments of diseased stems with a length of about 2-3 cm were washed under running tap water and then disinfected in a 70% ethanol solution for 1 minute. The alcohol was removed by washing three times with sterile distilled water, after which the collected fragments were dried with a sterile paper towel and 0.5 cm sections were laid out on plates with PDA medium and selective media for Phytophthora, PARP (Pimaricin, Ampicillin, Rifampicin, Pentachloronitrobenzene, Sigma-Aldrich, St. Louis, Missouri, USA) [28]. The cultures were incubated at 23 • C for 2-5 days and examined daily under a microscope microscope Eclipse 80i (Nikon, Tokyo, Japan). Fragments of growing mycelia indicating affiliation to the Phytophthora genus (hyphae branched almost at right angle, hyphal swellings, after Erwin and Ribeiro, 1996) [24] were laid on PDA medium for identification based on morphology or incubated in liquid organic medium for identification based on molecular markers. The re-isolation Agronomy 2020, 10, 1598 4 of 11 from the peat substrate was carried out using baiting of pathogen on leaves of the rhododendron cultivar Nova Zembla and blueberry, as in Section 2.3. Additionally, during the experiment, root fragments for isolation of P. cinnamomi were taken from plants with disease symptoms that were infected with the pathogen only and where both fungi were inoculated. At the termination of the experiment, root fragments were taken from all plants also without visible disease symptoms.

Assessment of Plant Growth and Tracking of Disease
At the beginning of the experiment, immediately before inoculation with S. indica, the number and length of all shoots of all plants was measured. After 24 months (September 2019), at the termination of the experiment, the same measurements were repeated and an increase in length was calculated for each living plant. Data were subjected to statistical analysis using one-way analysis of variance (Statistica v.13.1, Dell Inc., Round Rock, TX, USA), separately for each cultivar, and compared using a Tukey test at p = 0.05.

Colonisation of Roots with S. indica
S. indica was able to colonise roots of blueberry of the evaluated cultivars ( Figure 1). Chlamydospores, as a certificate of root colonisation, were present in each of the analysed plants but not in all root fragments. Percentages of inoculated roots were comparable between both terms of assessment, 9th and 24th month. Usually, fewer root fragments containing S. indica chlamydospores were found in plants inoculated with endophyte and pathogen than in those inoculated only with S. indica (Table 1). There were significant differences in colonisation frequency between cultivars. The lowest percentage of colonised roots was found in 'Spartan', which was susceptible to the pathogen and the highest in 'Darrow', which was the most resistant (Table 1). More chlamydospores indicate the possibility of more intensive secondary colonisation of newly formed roots, which enhances the protective potential of the endophyte.
Agronomy 2020, 10, x FOR PEER REVIEW 4 of 11 angle, hyphal swellings, after Erwin and Ribeiro, 1996) [24] were laid on PDA medium for identification based on morphology or incubated in liquid organic medium for identification based on molecular markers. The re-isolation from the peat substrate was carried out using baiting of pathogen on leaves of the rhododendron cultivar Nova Zembla and blueberry, as in Section 2.3. Additionally, during the experiment, root fragments for isolation of P. cinnamomi were taken from plants with disease symptoms that were infected with the pathogen only and where both fungi were inoculated. At the termination of the experiment, root fragments were taken from all plants also without visible disease symptoms.

Assessment of Plant Growth and Tracking of Disease
At the beginning of the experiment, immediately before inoculation with S. indica, the number and length of all shoots of all plants was measured. After 24 months (September 2019), at the termination of the experiment, the same measurements were repeated and an increase in length was calculated for each living plant. Data were subjected to statistical analysis using one-way analysis of variance (Statistica v.13.1, Dell Inc., Round Rock, TX, USA), separately for each cultivar, and compared using a Tukey test at p = 0.05.

Colonisation of Roots with S. indica
S. indica was able to colonise roots of blueberry of the evaluated cultivars ( Figure 1). Chlamydospores, as a certificate of root colonisation, were present in each of the analysed plants but not in all root fragments. Percentages of inoculated roots were comparable between both terms of assessment, 9th and 24th month. Usually, fewer root fragments containing S. indica chlamydospores were found in plants inoculated with endophyte and pathogen than in those inoculated only with S. indica (Table 1). There were significant differences in colonisation frequency between cultivars. The lowest percentage of colonised roots was found in 'Spartan', which was susceptible to the pathogen and the highest in 'Darrow', which was the most resistant (Table 1). More chlamydospores indicate the possibility of more intensive secondary colonisation of newly formed roots, which enhances the protective potential of the endophyte.

Influence of S. indica and P. cinnamomi on Growth of Plants
A positive effect of S. indica on the growth of shoots of the plants not infected with the pathogen was observed in 'Darrow' and 'Spartan' ( Table 2). The average increase in the length of all shoots of plants inoculated with S. indica was larger by 11% in 'Darrow' and 10% in 'Spartan' plants compared with non-inoculated plants of control. A similar relationship was recorded for the growth of the longest shoots; their increases were larger by 11% and 12% compared with the control that was not inoculated with the endophyte. Growth of the main shoots in 'Chandler' were completely independent of endophyte inoculation and even weaker in length of all shoots by 4% in plants inoculated with S. indica. One of the symptoms of blueberry infestation with P. cinnamomi was inhibition of plant growth. Inoculation of S. indica before P. cinnamomi infection reduced the negative effect of the pathogen on growth of blueberry plants (Table 2). This was particularly visible in 'Spartan'. The average growth increase of all shoots in combination 4 (S. indica + P. cinnamomi infection) was higher by 120% and in the length of longest shoots by 182% relative to combination 3 where plants were only infected with P. cinnamomi (Table 2). S. indica is known for its positive effect on the growth and development of plants, especially young plants, of many species, e.g., during acclimatisation of microplants produced in vitro [16,29]. In our experiments with rhododendron [30], S. indica significantly increased the height of shoots by 36% in 'Nova Zembla' and 13% in 'Alfred' after 7 months of the experiment and 33-36% after 15 months.

Influence of S. indica on Protection of Blueberry Plants against P. cinnamomi
The initial symptoms of infection with Phytophthora were visible as shoot necrosis in July 2018 (Figure 2), followed by diminutiveness, reddening, and dropping of leaves. Twelve months after inoculation with the pathogen, some infected plants were completely dry (dead). In the second year of the experiment, symptoms of phytophthorosis included continuous growth slowdown and formation of smaller diameter shoots (Figure 3). After 24 months, the number of plants visibly infected from which the pathogen was isolated differed between cultivars ( Table 2). The percentage of symptomatic plants inoculated only with P. cinnamomi was 59, 83, and 100% for 'Chandler', 'Darrow', and 'Spartan', respectively. In the treatment where both the endophyte and pathogen were used for inoculation, these percentages were 59, 28, and 90%, respectively. The highest severity of infection was found in 'Spartan', where 66% of plants that were not protected and 14% plants that were protected with S. indica died. In 'Chandler', 3 plants among the non-protected died and only one among the protected (out of 29). In 'Darrow', only one plant among the non-protected died, and none among the protected.

Influence of S. indica on Protection of Blueberry Plants against P. cinnamomi
The initial symptoms of infection with Phytophthora were visible as shoot necrosis in July 2018 (Figure 2), followed by diminutiveness, reddening, and dropping of leaves. Twelve months after inoculation with the pathogen, some infected plants were completely dry (dead). In the second year of the experiment, symptoms of phytophthorosis included continuous growth slowdown and formation of smaller diameter shoots (Figure 3). After 24 months, the number of plants visibly infected from which the pathogen was isolated differed between cultivars ( Table 2). The percentage of symptomatic plants inoculated only with P. cinnamomi was 59, 83, and 100% for 'Chandler', 'Darrow', and 'Spartan', respectively. In the treatment where both the endophyte and pathogen were used for inoculation, these percentages were 59, 28, and 90%, respectively. The highest severity of infection was found in 'Spartan', where 66% of plants that were not protected and 14% plants that were protected with S. indica died. In 'Chandler', 3 plants among the non-protected died and only one among the protected (out of 29). In 'Darrow', only one plant among the non-protected died, and none among the protected.  The severity and course of Phytophthora infection on blueberry was different than that described for rhododendron [21]. Rhododendron plants showed symptoms soon after infection. Within one to two weeks, leaves were bent, wilted, and dried quickly, and all plants died within six weeks in greenhouse experiments [21]. The severity of disease was similarly strong in two cultivars. In contrast, the blueberry plants that were infected at the age of 6 months endured the disease more easily, symptoms were visible later, and only some of them died, although Phytophthora was isolated from the shoots, roots, and substrate. The symptoms of disease on plants that did not die within 2 years of the experiment included stunted growth and development of thinner shoots compared to plants not infected with the pathogen. Root balls were smaller in volume and darker in colour (Figure 4). In contrast to rhododendron [21], the cultivars of highbush blueberry differed in resistance to P. cinnamomi. The most susceptible was 'Spartan' and the most resistant was 'Darrow'. To our surprise, the leaves of 'Chandler' and 'Darrow' were resistant to infection with P. cinnamomi spores (they could not capture the pathogen from the substrate) and because of this, it was not possible to use them in baiting for re-isolation of the Agronomy 2020, 10, 1598 7 of 11 pathogen from the substrate. Only leaves of the most susceptible cultivar, Spartan, were able to be infected with the pathogen (Figure 5). Bryla [9] studied the incidence of Phytophthora and Pythium infections on the blueberry fields of Oregon but did not find symptomatic plants of 'Darrow' among the eleven monitored cultivars. The severity and course of Phytophthora infection on blueberry was different than that described for rhododendron [21]. Rhododendron plants showed symptoms soon after infection. Within one to two weeks, leaves were bent, wilted, and dried quickly, and all plants died within six weeks in greenhouse experiments [21]. The severity of disease was similarly strong in two cultivars. In contrast, the blueberry plants that were infected at the age of 6 months endured the disease more easily, symptoms were visible later, and only some of them died, although Phytophthora was isolated from the shoots, roots, and substrate. The symptoms of disease on plants that did not die within 2 years of the experiment included stunted growth and development of thinner shoots compared to plants not infected with the pathogen. Root balls were smaller in volume and darker in colour (Figure 4). In contrast to rhododendron [21], the cultivars of highbush blueberry differed Diseased plants are characterised by lower productivity but will die under adverse conditions, such as flooding or drought. Symptoms of phytophthorosis on field grown plants look similar to those observed in our greenhouse trial and were characterised with "small, yellow or red leaves, lack of new growth, root necrosis, and a smaller root system, and they gradually died within three years, especially when grown in wet soil" [10]. In older plants, the disease could be chronic and diseased plants were less productive, including the quality and quantity of yield.

Re-Isolation of Pathogen from Plants and Substrate
Our trials on baiting the pathogen from the substrate on leaves resulted in colonisation of rhododendron and blueberry 'Spartan' leaves (Table 3, Figure 5). Baiting was successful from all substrate samples inoculated with P. cinnamomi and in both terms of investigation. The number of necrotic spots showed the amount of pathogen propagules and was the highest in samples where 'Spartan' was grown (Table 4). This can be a starting point to develop a test for the preliminary screening of blueberry genotypes for resistance to this pathogen. Diseased plants are characterised by lower productivity but will die under adverse conditions, such as flooding or drought. Symptoms of phytophthorosis on field grown plants look similar to those observed in our greenhouse trial and were characterised with "small, yellow or red leaves, lack of new growth, root necrosis, and a smaller root system, and they gradually died within three years, especially when grown in wet soil" [10]. In older plants, the disease could be chronic and diseased plants were less productive, including the quality and quantity of yield.

Re-Isolation of Pathogen from Plants and Substrate
Our trials on baiting the pathogen from the substrate on leaves resulted in colonisation of rhododendron and blueberry 'Spartan' leaves (Table 3, Figure 5). Baiting was successful from all substrate samples inoculated with P. cinnamomi and in both terms of investigation. The number of necrotic spots showed the amount of pathogen propagules and was the highest in samples where 'Spartan' was grown (Table 4). This can be a starting point to develop a test for the preliminary screening of blueberry genotypes for resistance to this pathogen. Other authors also reported differing susceptibility of blueberry cultivars to Phytophthora [11,31]. In greenhouse conditions, 12-or 18-month-old plants were inoculated and the level of resistance was evaluated on the basis of shoot and root biomass [31] or growth parameters and fresh weight [11] after 16 (the first report) and 13 (the second) weeks. Within this time, no dead plants were observed. In our experiment, some plants died within 2 years. According to Smith [13] and Bryla [9], Phytophthora diseases of blueberry in productional fields can have a chronic character. Diseased plants are characterised by lower productivity but will die under adverse conditions, such as flooding or drought. Symptoms of phytophthorosis on field grown plants look similar to those observed in our greenhouse trial and were characterised with "small, yellow or red leaves, lack of new growth, root necrosis, and a smaller root system, and they gradually died within three years, especially when grown in wet soil" [10]. In older plants, the disease could be chronic and diseased plants were less productive, including the quality and quantity of yield.

Re-Isolation of Pathogen from Plants and Substrate
Our trials on baiting the pathogen from the substrate on leaves resulted in colonisation of rhododendron and blueberry 'Spartan' leaves (Table 3, Figure 5). Baiting was successful from all substrate samples inoculated with P. cinnamomi and in both terms of investigation. The number of necrotic spots showed the amount of pathogen propagules and was the highest in samples where 'Spartan' was grown (Table 4). This can be a starting point to develop a test for the preliminary screening of blueberry genotypes for resistance to this pathogen. Table 3. Re-isolation of P. cinnamomi from substrates inoculated with P. cinnamomi on which blueberry plants were grown. Re-isolation was made by baiting on leaves of rhododendron (RH) and highbush blueberry (HB); success (+); lack (−).  The idea of bio-protection of this crop was attempted in experiments by De Silva [32] that aimed to promote growth using several beneficial microorganisms. Only the fungus Gliocladium virens Gl. 21 and bacteria Pseudomonas fluorescens PF 5 promoted growth of 'Blueberry'. The experiment demonstrated the usefulness of G. virens to increase blueberry growth in a nursery. Our results are the first on bio-protection of highbush blueberry against pathogens of Phytophthora spp. using the endophytic fungus S. indica and may be an introduction to the development of technology for use in blueberry nurseries.

Cultivar
The most important guideline for effective biological protection of blueberries is to know the mechanism of plant's natural defences against Phytophthora root rot, as well as how S. indica enhances the plant's resistance. So far, some elements of the resistance mechanism to Phytophthora spp. have been determined for a few trees and shrubs, but they cannot be considered sufficient for generalisation [33]. Although the S. indica endophyte has recently come to be better understood, there is still a lack of complete knowledge about the general mechanism of stimulating resistance in plants [34]. It seems that this fungus very "intelligently" uses the genetic possibilities of the host plant to build a safe niche for itself. In the coming study, we will try to elucidate this issue.

Conclusions
We can conclude that in the greenhouse pot experiment, S. indica protected highbush blueberry plants against severe phytophthorosis; thus, counteracting growth retardation and reducing the number of symptomatic and dead plants. Highbush blueberry cultivars differ in their sensitivity to P. cinnamomi. The introduction of S. indica into the production of highbush blueberry planting material may have the potential to reduce the chemical protection of this plant against P. cinnamomi.