Role of Endophytes in Apple Replant Disease

Abstract

Apple replant disease (ARD) is a major problem in all the apple-growing areas of the world. It is a complex problem. The exact cause of the problem is unknown, but soil biotic factors play a major role. The repeated cultivation of same crop on same land and exhaustion of nutrients of soil, persistence of soil-borne pathogens and changes in the pH of soil. Symptoms include stunting of tree growth with short internodes, small and light green rosette leaves, development of few lateral or feeder roots, underdeveloped root systems, decayed and discolored roots, poor establishment and severe disease results in the death of young trees and, sometimes, whole orchards. The endophytes provide direct benefits to host plants as they live in close proximity. Once they enter inside the host tissue they get easily established as they feel no competition with other microorganisms. Endophytes have the capacity to produce different secondary metabolites, which saves the host plants from biotic and abiotic stresses the host plants become resistant to both biotic and abiotic stresses. An interesting facet of the interaction between endophytes and their hosts is the capacity of many microorganisms to improve the plant’s resistance by providing several bioactive metabolites. Therefore, the exploitation of soil microbial endophytes for the management of ARD is an important strategy.

1. Introduction

The apple replant disease (ARD) is widespread throughout the world, and severity of this disease varies from region to region, fields, type of soil and presence of pathogenic microbial species in the rhizosphere. This problem may occur due to repeated cultivation of same crops on the same land and exhaustion of nutrients of soil, persistence of soil-borne pathogens and changes in the pH of soil. Replant disease is a debilitating soil problem affecting most orchards when they are replanted. This problem affects plants worldwide both in the nurseries, as well as in the orchards, in terms of plant growth, yield and quality of fruits [1, 2]. Different authors give different definitions of ARD. The main cause of apple replant disease is the imbalance in the soil microbial community [3], but the fungal pathogens vary from region to region. According to Utkhede [4], replant problem is caused by both biotic and abiotic factors, which suppress the growth of plants, whereas the replant disease is only caused by the biotic community present in the rhizospheric soil. Savory [5] defines ARD as soil sickness whose causes are unknown and uncertain. This problem is seen in other commercial crops, such as peach, pear, cherry, strawberry and rose.

Apple replant disease (ARD) affects the plant nurseries, as well as apple production worldwide by strongly reducing plant growth as well as fruit yield and quality [2, 3]. On ARD soils, over the lifetime of an apple orchard, a 50% reduced profitability has been estimated due to later and less fruit-bearing of the affected trees [6, 7]. ARD becomes problematic nowadays as the tree nurseries and fruit orchards are concentrated in certain regions of Pinneberg in Germany or Pistoia in Italy. Also, due to high-density plantations in apple crops, farmers are using dwarf rootstocks to achieve more yield in a short life span, which results in frequent replantation of apple orchards [8, 9]. Replant problem is very much severe and its control is very difficult. Crop rotation is the only method to reduce the effect of replantation, but it is also difficult due to alternative usage for industry, energy plants or other purposes. Different authors gave different definitions of the term ‘replant disease’ or related phrases, such as ‘replant problem’, ‘soil sickness’ or ‘soil fatigue’ [4, 10, 11]. These biotic and abiotic factors of replant problem suppress plant growth [4]. The unknown and uncertain cause of reduced growth is known as ‘soil sickness’ [5], which excludes nematode damage [12]. ARD is specifically related to species Malus domestica, and its persistence for decades is noticed [5]. The impact of ARD is reversible when plants are transplanted into virgin or healthy soil. This disease has been reported in other crops, such as rose, cherry, peach, strawberry and rowan, whereas roses are prone to it. Here we want to summarize the causes of ARD and their mitigation strategies. The apple trees may grow poorly when planted in non-sterilized soil. The problem occurs when same crop is planted in the same land year after a year, which disturbed the root microflora of the plants due to disturbed physiological and morphological reactions of apple plants to soil [13].

There are two forms of replant diseases: specific and non-specific. Specific replant disease only affects apple crops when the site is again replanted with the same crop, whereas non-specific replant disease occurs when stone fruits are planted on the site where previously planted with apple crops. The root cause of non-specific replant disease is nematode activity and fungal pathogens on the replanted sites. It was confirmed by using a nematicide pot test that 40% of Tasmanian apple orchards have both specific and non-specific replant disease (HAL project AP97005). It is a complex problem caused by number of pathogenic organisms, such as actinomycetes (filamentous bacteria), nematodes and bacteria, and fungi, such as Rhizoctonia, Pythium and Phytophthora. This has led to the conclusion that there is no specific treatment to control this disease with less impact on beneficial soil organisms. Soil sterilants, such as methyl bromide and chloropicrin, are used to kill the pathogenic microflora. Nowadays, there are eco-friendly approaches, such as organic matter, fertilizers, microorganisms, irrigation, cultivation and rootstocks, which have been used, but none of these are 100% effective to control the replant disease [14, 15, 16].

Apple replant disease is characterized by a severely reduced rate of root and shoot growth of second planting of same species or closely related species on same site, whereas non-specific causes of apple replant disease (ARD) mean incorrect use of fertilizers, poor soil structure, poor drainage, pH imbalances and presence of toxic compounds, such as herbicides, heavy metals and biological products. The main cause of ARD in New York apple replant orchards were nematodes (Pratylenchus penetrans), parasitic fungi, bacteria and other soil-borne microorganisms. The possible abiotic factors reported in the replant sites were poor nutrient availability, change in the pH of the soil, deteriorated soil structure and loss of organic matter, herbicide residues and other site-specific problems [17]. In Washington state soils, the Pratylenchus penetrans number was below the damage threshold level in eight of the nine orchards surveyed and bacteria were not identified in the disease. The main fungal pathogens associated with the disease were Phytophthora cactorum, Pythium spp., Cylindrocarpan destructans and Rhizoctonia solani [6].

Himachal Pradesh is known as ‘apple state’ of India as its cultivation has revolutionized the socio-economic status of the farmers and had played a pivotal role in the economy of growers. Apple orchards planted in late sixties have shown symptoms of declining productivity as these plants have completed their life span. Due to less land resources and choice of apple crop, old apple orchards are replanted with the same crop, which leads to drastic economic losses due to uprooting of old trees and poor establishment of new plantations on the replant site. The continuous cultivation of the same crop on the same field is the primary factor leading to replant problem. Due to this, a general decline in the growth and productivity of replanted apple orchards is commonly observed. The replant disease is one of the most important diseases in newly established orchards in old orchard sites of Himachal Pradesh [18]. This disease is very common in other stone fruit crops, such as cherry, strawberry, peach and pear, in Himachal Pradesh.

1.1 Symptoms

The disease symptoms of ARD are visible within 1 year of plantation. Disease symptoms affect the whole orchards, which include poor and uneven growth of trees and yield in future [10, 19]. It is very difficult to predict whether replant problem is present on a specific site or not. It is also difficult to assume that rootstock or nursery plants are the root cause for poor tree performance of apple trees. Replant disease affects most fruit crops including both pome and stone fruits.

Symptoms include stunting of tree growth with short internodes, small and light green rosette leaves, development of few lateral or feeder roots, underdeveloped root systems, decayed and discolored roots, poor establishment and severe disease results in the death of young trees and, sometimes, whole orchards. When a tree is uprooted, discolored roots, root tip necrosis and reduced root biomass can be seen. The cell wall degrading enzymes and cell-killing effector proteins, produced by pathogenic micro and macroflora, destroy the root tissues of host plants [20, 21, 22, 23, 24]. It impairs root function hampering plant responses to abiotic stresses, such as drought, flooding and nutrient deficiency.

1.2 Disease etiology

The apple replant disease is caused by number of pathogenic microflora and microfauna. The etiology of the disease varies from region to region and site to site. It depends on the dead tissues of the plant’s remains in the soil, which insist the soil to attract the pathogenic microflora towards them. The ARD in Washington State is caused by different pathogenic fungi, such as Rhizoctonia, Ilyonectria; oomycetes: Phytophthora, Pythium and in some sites lesion nematode Pratylenchus penetrans [6]. The contaminated soil, irrigation water or planting stocks are different sources of inoculums to spread the ARD. The pathogenic fungi produce overwintering structures, such as sclerotia (Rhizoctonia), chlamydospores (Ilyonectria) and oospores (Phytophthora and Pythium), survive in dead or dormant roots. Lesion nematodes survive in the soil and plant residues as eggs and as multiple generations of juveniles and adults [25]. During the spring season, when new apple plants are planted on the same site, the active dormant roots secrete root exudates, which help the pathogen propagules to germinate and parasite nematode eggs to hatch. The propagules germinate to produce the fungal and oomycete mycelia and then infect the new fine roots. While fungi such as Rhizoctonia cause extensive root rot, Pythium spp. cause damping off in the root tissue resulting in diminished capacity of the plant to take up water and nutrients. The different causal agents of ARD, such as Phytophthora cactorum, Pythium ultimum, Fusarium oxysporum and Dematophora necatrix, were isolated from the temperate zones of Himachal Pradesh [18]. The primary cause of the disease is the over-exploitation of the old apple orchards specialized in fruit production. The causes of replant problem are not exactly clear but it is a complex problem and occurs mainly due to disturbances in the healthy microflora of soil, variation in soil pH, organic matter, root exudates of old tree roots, water retention in the rhizosphere area and plant genotypes [13].

Fungi of the genera Cylindrocarpon, Rhizoctonia, Phytophthora and Pythium are found frequently in ARD-affected soils and have proved to be crucial in the etiology of ARD [3, 18, 26]. Different Fusarium spp., such as F. oxysporum, F. solani, F. equiseti and F. proliferatum, were isolated from 10 different apple nurseries in Tunisia, which causes considerable losses to apple plants. Root endophytic Cylindrocarpon-like fungi Thelonectria sp. and Ilyonectria spp. were also associated with ARD [23], after Pythium spp., to be correlated to the growth reduction in the rootstock M9 growing in ARD-affected soil. Different species of Nectriaceae were also found in ARD-affected cortex cells applying laser microdissection [27]. Several fungal endophytes from ARD-affected apple roots were isolated and re-inoculated in a soil-free biotest [28].

Several Streptomyces amplicon sequence variants (ASVs) were detected in greenhouse ARD biotest, which were negatively correlated to shoot length and fresh mass, from both field sites. The Streptomyces ASVs in roots of apple plants in control soil increased their relative abundance over time. The 150 bacterial strains isolated by a culture-dependent approach revealed a high diversity of members of the genus Pseudomonas, confirming the data of the molecular barcoding approach. Therefore, it is important to combine these two approaches to better understand this complex disease and develop control measures. Finally, Streptomyces play a key role in the etiology of ARD in the study sites [29].

2. Mechanism of plant prophylaxis by endophytic microorganisms

The plant-associated microorganisms are important in reference to safety and quality of fruits. There are two types of soil microorganisms, rhizospheric and endophytic soil microorganisms. Both types of microorganisms have the same functions. The main difference between these two is, once the endophytic microorganisms enter the host tissue, they are protected from the changing environmental vagaries [30]. They provide direct benefits to host plants as they live in close proximity. Plant growth-promoting endophytic bacteria affect the plant growth through two mechanisms, such as direct and indirect ways [31]. The direct mechanisms include production of growth hormones (auxins, cytokinins and gibberellins), phosphate solubilization, siderophore production, competition and lytic enzymes secretions, whereas the indirect mechanisms of growth promotion include induction of plant resistance, predation and hyperparasite and production of antifungal metabolites, cell wall degrading enzymes, decreasing the amount of iron available to phytopathogens and synthesis of pathogen-inhibiting volatile compounds [32].

3. Exploitation of microbial endophytes as ARD management strategy

The word endophyte means ‘in the plants’. These are isolated from internal tissues of plants by following the proper sterilization procedures. Microbial associations with host plants may be epiphytic, parasitic, mycorrhizal, endophytic, saprophytic etc. Only the epiphytic and endophytic microorganisms make their way to internal tissues of host plants. Endophytes include symbiotic associations of bacteria, fungi and yeast with the host plant [33]. Both partners are equally benefitted from each other. Many endophytes are members of common soil bacterial genera, such as Bacillus, Pseudomonas, Burkholderia and Bacillus [34]. Both the bacterial and fungal endophytes are present in the plant tissues without causing any ill effects. As the endophytes live inside the host tissue, they are beneficial to the host plants because they improve host plant tolerance to abiotic stresses, enhance growth, improve plant immune response and suppress pathogen colonization [35].

In comparison to rhizospheric microorganisms, endophytic microorganisms are more beneficial because they live in close proximity to host plants and exert direct benefit to host plants. Also, they live in non-competitive environments [36]. Endophytes improve plant growth by secreting phytohormones and consequently help in nutrition improvement using bidirectional nutrient transfer and enhancement of the health of plants by protecting them against phytopathogens [37, 38].

Endophytic bacteria are diverse in nature, most commonly represented by the genera Pseudomonas, Bacillus, Burkholderia, Stenotrophomonas, Micrococcus, Pantoea and Microbacterium [31]. The beneficial functions of well-known bacterial endophytic genera Chitinophaga and Flavobacterium in plant interactions in less known [39]. The most widely used genus, Bacillus has beneficial functions in its interactions with agriculturally-important plants. The endophytic bacteria indirectly promote plant growth by secreting antifungal metabolites or stimulating plant defense responses. The plant defense response, such as induced systemic responses (ISR), acts through specific plant response pathways, such as jasmonic acid (JA) pathway [40], but recently endophytic bacteria use the signalling pathways to stimulate these defense responses.

Endophytic fungi internally colonize plant tissues without causing any harm to the host plant [41]. The large group of endophytic fungi are commensals, some are mutualists [42, 43]. The most commonly exploited endophytic fungi belong to genera Aspergillus, Bipolaris, Chaetomium, Cladosporium, Diaporthe, Fusarium, Alternaria, Mucor, Nigrospora, Paecilomyces, Penicillium, Piriformospora, Porostereum, Phoma, Trichoderma, Ulocladium and Yarrowia [44, 45].

Therefore, the exploitation of these resident soil microbial communities for the management of ARD is a better strategy as plant-beneficial microflora helps in the establishment of new plants planted in old orchards sites and simultaneously increases the innate immunity of host plants by secreting bioactive metabolites. As we know that the bacteria on roots and in the rhizosphere is benefitted from plant root exudates. But it is not clear which population is more advantageous for the plants in terms of establishment of plants, growth, survival and yield. Endophytic populations like rhizospheric populations are conditioned by both the biotic and abiotic factors, but endophytes could be better protected from both the stress factors.

Exploitation of endophyte–plant associations result in the promotion of plant health and can play a significant role in low-input sustainable agriculture practices for all crops. By using molecular approaches, the whole genome sequences of key endophytic bacteria are available and the genes used in colonization and establishment of endophytic bacteria in plants can be identified.

Trichoderma asperellum strain 6S-2, an effective fungal endophyte isolated from roots of healthy apple trees growing in nine replanted orchards in Shandong Province, China, showed different lytic activities, such as protease, amylase, cellulase and laccase, which are important for the parasitic and antagonistic functions of pathogenic fungi. The inhibition rate of this fungal strain 6S-2 against phytopathogen Fusarium proliferatum f. sp. M. domestica MR5 was 52.41%. Strain 6S-2 also secreted different plant growth promoters, such as iron carriers, auxin and ammonia, and was able to solubilize phosphorus. The fermentation extract and volatile substances produced by endophyte Trichoderma asperellum inhibited the growth of Fusarium proliferatum f. sp. M. domestica MR5, by twisting, shrinking, swelling and rupturing the pathogen hyphae associated with apple replant disease [46].

Endophytic bacteria are able to lessen or prevent the deleterious effects of certain pathogenic organisms to host plants. The beneficial effects of bacterial endophytes on their host plant appear to occur through similar mechanisms as described for rhizosphere-associated bacteria. Bacterial endophytes may have an advantage over bacteria inhabiting the rhizosphere since living within a plant’s tissues represents an opportunity to always be in contact with the plant’s cells and therefore to more readily exert a direct beneficial effect. Of course, bacteria residing in the rhizosphere might also have the potential to enter and colonize the plant roots.

The maximum mycelial inhibition of 81.48% was obtained with the fungal endophyte Aspergillus aculeatus strain C2 under in vitro conditions. Microscopic studies on interaction between fungal endophytes with hyphal tips of apple root rot pathogen Dematophora necatrix revealed various morphological abnormalities in the hyphae like curling and bending of mycelium. Under glasshouse conditions, seed treatment pursued by soil application with fungal endophyte Crinipellis tabtim strain M8 isolate was highly effective and exhibited 93.55% disease control. Similarly, under field conditions, the overall maximum disease control was exhibited by Crinipellis tabtim strain M8 (84.95%). The most promising root endophytes were identified on morphological and ITS sequence analysis. Root colonization assay revealed maximum endosphere and rhizosphere colonization with Crinipellis tabtim strain M8. Also, confocal microscopic illustrations of transverse sections of root cells tenanted by fungal endophytes Crinipellis tabtim strain M8 as compared to untreated control suggested the persistence and establishment of endophytes in the endosphere of apple seedlings, shown in Figure 1 [47].

Figure 1.

Microscopic elucidation of colonized fungal root endophytes. (a) Crinipellis tabtim strain M8. (b) Control by confocal laser scanning microscopy (CLSM by Pal et al. [47]).

3.1 Helps in the establishment and proper growth of new apple plantation

Endophytes have the capacity to secrete different secondary metabolites, such as phenolic acids, alkaloids, quinones, steroids, saponins, tannins and terpenoids, which help the host plants to resist both biotic and abiotic stresses. The important secretions by these endophytes helped them in anticancer, antimalarial, antituberculosis, antiviral, antidiabetic, anti-inflammatory, anti-arthritis and immunosuppressive roles. The interaction between endophytes and their hosts is the capacity of many microorganisms to improve the plant’s resistance by providing several bioactive metabolites [48]. The secretion of volatile compounds by plant-associated bacteria in association with host plants is a promising sustainable strategy to prevent the soil-borne and foliar fungal pathogens [49, 50, 51]. Plant-associated mutualistic microorganisms such as endophytic microorganisms, commonly known as endophytes, which colonize plants’ internal tissues, frequently contribute to host metabolic function and protect plants against pests and diseases by producing biocontrol traits, such as bioactive secondary metabolites. The bacterial endophytes generally colonize the internal tissue of plants and are found nearly in every plant. The mechanism followed for plant growth promotion by some of the bacterial endophytes is same as that followed by rhizosphere bacteria. Inoculation of part of a plant with an endophyte may benefit plants via the production or suppression of phytohormones; for example, genes encoding proteins for biosynthesis of indole acetic acid (IAA), cytokinins (CKs) and gibberellins (GAs) are often present in the metagenome of plant endophytic bacterial communities. The application of spore suspension of Trichoderma asperellum strain 6S-2 to replanted apple orchard soils reduced plant oxidative damage and promoted plant growth in a pot experiment and the strain 6S-2 demonstrated plant growth-promoting activities such as protease, amylase, cellulase and laccase activities, which are important for the parasitic and antagonistic functions of pathogenic fungi. The inhibition rate of 6S-2 against Fusarium proliferatum f. sp. M. domestica MR5 was 52.41% and thus helps in the establishment of the young planatations to the replanted sites [13]. Compared to rhizosphere soil strains, endophytic microorganisms can effectively colonize host plants and better adapted to environmental changes, making them more effective in disease suppression and growth promotion [52]. The culture-independent and culture-dependent approach used in 3, 7 and 12 months planted apple plants in ARD-affected and ARD un-affected soil at two sites reported that a high diversity of Pseudomonas in all soils and by using molecular bar-coding approaches an increase in relative abundance of Actinobacteria in plants grown in ARD and control plots [29].

3.2 Helps in disease suppression

Microbial endophytes live in association with the host plants without causing any side effects but their presence is beneficial to the host plant, as they protect the plants from biotic and abiotic stresses simultaneously enhance the growth and modulate plant immune response and suppress the pathogen colonization [35]. Endophytes reduce the negative impact of pathogens on their host by secreting siderophores, antibiotics, cell wall degrading enzymes, volatile organic compounds (VOCs), alkaloids, steroids, quinines, terpenoids, phenols and flavonoids, which are inhibitory to the phytopathogens [53, 54], or by interrupting the cross-talk signal of pathogens [55]. Secretion of lytic enzymes (β-1,3-glucanase, chitinase, cellulase and protease) by endophytes helps in degrading the cell wall of pathogenic fungi. Chitin, a major cell wall component of fungi, is degraded by enzyme chitinase. Chitinase produced by endophytic Streptomyces hygroscopicus, inhibit the growth of pathogenic fungi Ralstonia solani, Fusarium oxysporum, Alternaria alternata, Aspergillus niger, Aspergillus flavus, Sclerotinia sclerotiorum, Hyaloperonospora parasitica and Botrytis cinerea [56]. Endophytes help in plant growth either by producing secondary metabolite or nutrient assimilation or by preventing induction of plant disease symptoms by different pathogens. Endophytes produce a large number of bioactive compounds, which provide resistance to host plants and simultaneously protect the plants from biotic and abiotic stresses [57].

3.3 Increase the innate immunity of host plants

The bacterial endophytes enter the host system by following the same mechanisms as used by the bacterial pathogens, but the host plant allows the entry of specific genera to colonize the inner host tissues. Due to this, close interaction brings important changes in the plant physiology. The bacterial symbiont living in association with host exerts direct and indirect defenses to control plant biotic stresses. These defenses may be due to the secretion of volatiles and antibiotic compounds by endophytes, therefore, boosting the innate immunity of host plants against various diseases [58]. The plants allow the microbial colonization through phenotypic genes and producing metabolic signals. Host plants by evolving their genotypes provide sugar and lipids to the endophytes. Therefore, a plant’s genotype can influence the microbiome composition and shape microbiome to enhance defense and mitigate the trade-off between growth and defense against pathogens by secreting chemoattractants [59].

4. Conclusions

Endophytes help in plant growth either by producing secondary metabolite or nutrient assimilation or by preventing induction of plant disease symptoms by different pathogens. The application of these endophytes may increase the availability of nutrients and control the replant disease organisms and considerably will regenerate, maintain and sustain the soil fertility and hence the establishment of apple rootstocks and yield in future. Therefore there is an urgent need for isolation, identification and characterization of indigenous plant growth promoting endophytes associated with apple plants as plant growth promoter in the fields as bioformulations. This will help to solve the replant problem of apple orchards and improve the economy of state.

Acknowledgments

We are highly thankful to the Department of Science and Technology, GOI, New Delhi for providing financial assistance under the grant no. DST/WOS-B/2018/1833.