Fusarium spp. associated to citrus dry root rot: an emerging issue for Mediterranean citriculture

Fusarium root rot and dry rot are destructive diseases of citrus trees that gradually affect plants under biotic or abiotic stress. Many Fusarium spp. are supposed to be the casual agents of these diseases and their identification could be of an extreme difficulty. In this work, Fusarium spp. were collected from commercial citrus orchards and nurseries in Italy, Tunisia, Greece and Egypt. Three Fusarium species (F. solani, F. oxysporum and F. proliferatum) were identified according to morphological and molecular characterization, thus F. solani and F. oxysporum were the most frequent isolated species and, accordingly, were grouped upon the sequence of β-tubulin and α-Elongation Factor loci into nine clusters. Three pathogenicity characters were assessed: phytotoxicity of fungi excretions, capacity to colonize the surface of citrus host plant and ability to colonize the internal tissues. All trials were carried out on citrus, ‘Carrizo’ citrange. Symptoms of wilting were assessed over two months in phytotoxicity of culture filtrate trial. The results highlighted a significant correlation between phytotoxicity excretions and invasion ability. Nevertheless, no correlation was found between these two measures and the ability to colonize the surface of citrus seedlings. Despite the large variation existing among strains within the species, strains belonging to F. oxysporum showed a higher aggressiveness among all the characters tested, thus confirming the previous results obtained on the pathogenicity of F. oxysporum strains and F. sp. citri. Due to the fact that all the tested isolates were collected from orchards known to have dry root rot symptoms, and according to our finding regarding the higher aggressiveness of F. oxysporum isolates on citrus seedlings, a correlation might be suggested between Fusarium wilt disease affecting citrus seedlings and dry root rot disease observed on scaffold roots of trees in the orchards. INTRODUCTION Fusarium spp. are commonly found in soils of citrus orchards (Sherbakoff, 1953; Le Roux, 1985; Nemec et al., 1989b) and of nurseries (Wehner et al., 1987). The main Fusarium species isolated from citrus is F. solani (Menge and Nemec, 1997); however, other Fusarium species have been isolated from citrus; F. oxysporum was isolated from the roots and rhizosphere, but its isolation frequency was lower than the one of F. solani (Labuschagne et al., 1987; Nemec et al., 1989a). F. equiseti have been rarely isolated from citrus roots in Florida, their potential role as pathogens is still unknown (Smith et al., 1988). Recently, Leslie and Summerell (2006) reported that F. equiseti and F. semitectum are saprophytic colonizers regularly found as secondary invaders of diseased tissues. In citrus orchards in Greece, three species of Fusarium were isolated from roots: F. solani, F. proliferatum and F. sambucinum; they were found to act as pathogens on artificially inoculated potted plants, especially under conditions of water stress, excessive fertilization or root wounding (Malikoutsaki-Mathioudi et al., 1987). There are three diseases reported on citrus and supposed to have relation with Fusarium spp.: (i) citrus dry root rot; (ii) citrus blight; and (iii) Fusarium wilt of citrus seedlings. Fusarium dry root rot is a sporadic but destructive disease, occurs in citrus orchards that are usually affected by some other factors, biotic like: Phytophthora spp. (Dandurand and Menge, 1992) and nematode infections (O’Bannon et al., 1967; Proc. XXVIIIth IHC – IS on the Challenge for a Sustainable Production, Protection and Consumption of Mediterranean Fruits and Nuts Eds.: A.M. D’Onghia et al. Acta Hort. 940, ISHS 2012 648 Sherbakoff, 1953), or abiotic like: drought, water logging, temperature (Nemec and Zablotowicz, 1981), mechanical root injury, excessive nitrogen, salinity, etc.). Symptoms are similar to those caused by Phytophthora but could be distinguished as they usually appear as decay of larger roots and trunk below the bud union without oozing any gum, i.e., dry rot. In addition, sometimes the wood below the bark is discolored, while canopy exhibit general weakness and reduced vigor, leaves appear dull green, restricted new growth and twig dieback (Sakovich, 2003). The disease can be symptomless for years, but once enough root tissue has been destroyed, sudden collapse can occur under dry hot conditions. Citrus blight is also an important decline disease of citrus in many countries. The disease is characterized amongst other symptoms, wilt and dieback of the canopy, reduced water uptake and zinc accumulation in the trunk wood. F. solani was implicated by Baker et al. (1981) and Nemec et al. (1980) as a possible causal agent of citrus blight. Trees affected with dry root rot generally decline quickly within a year or two of first developing symptoms. Fusarium solani caused fibrous and scaffold root rot are the only potential disease symptoms linked to blight, and they are identical to the symptoms on dry root rot diseased trees (Menge and Nemec, 1997). Both diseases have been associated with climatic and edaphic predisposing factors. All rootstocks are susceptible to dry root rot and blight, but rough lemon is the most susceptible to both diseases (Bender, 1985; Nemec and Myhre, 1992). Studies by Nemec et al. (1980) and Labuschagne and Kotze (1988) showed that the fungus causes on inoculated seedlings symptoms that are identical to those on field trees. On adult plants, the most conspicuous symptom above the soil level is a fatal collapse of the tree (Ippolito and De Cicco, 1995) leaves suddenly wilt and dry up in a few days, remaining attached on the tree. Normally, the course of the disease is chronic and symptoms look like those provoked by other root rot agents: trees show a lingering decline, progressing for years, with the only initial symptoms being a slight wilt under dry conditions and a poor vegetation flush (Polizzi et al., 1992). At more advanced stage of the decline, the canopy has a thin and sparse appearance. In some cases, there is also the production of an unusually high number of fruits, stemming from a delayed bloom. F. solani is a facultative parasite, or in other words, an opportunistic pathogen. Opportunities to expand infection into other tissues besides the epidermis may be triggered by the interaction with other citrus root pathogens (Bender, 1985; Fawcett, 1923; Menge et al., 1981; O’Bannon et al., 1967; Van Gundy and Tsao, 1963), or by edaphic, cultural and other environmental factors. Stress may be important in predisposing the citrus plant to infection and disease caused by Fusarium spp. (Dandurand and Menge, 1992). Although, in the absence of stress, mild isolates of the fungus are able to colonize healthy roots establishing a symptomless infection (Nemec, 1978). The presence of the fungus in symptomless roots suggests that it could be a potential cause of symptoms in trees, especially in stressed plants. Moreover, the infection can start from the nursery and progress for many years without inducing any symptoms, but under stress conditions the disease can develop in the field. In the Mediterranean basin, most citrus varieties are grafted on sour orange rootstock. Therefore, the development of dry root rot is quite uncommon because sour orange is generally tolerant to stresses (Bender, 1985). Recently, the replacement of this rootstock should be considered to prevent the possible spread of Citrus tristeza virus (CTV). Thus, a survey should be anyway carried out in nurseries and fields where the fungus can induce symptomless infections. This study aimed to survey Fusarium spp. populations in citrus nurseries and groves in Italy, Greece, Tunisia and Egypt, then the identification and purification of the collected isolates aimed to study the correlation between the collected isolates using the phylogeny analyses basing on β-tubulin (tub-2) and α-Elongation Factor (tef1) loci sequence. Furthermore pathogenicity characters for selected 13 isolates of Fusarium spp. were evaluated on ‘Carrizo’ citrange seedlings in two in vitro trials.