Relative susceptibility of citrus genotypes to fruit rot caused by Ceratocystis radicicola in Iran

SHORT COMMUNICATION COMUNICAÇÃO

Relative susceptibility of citrus genotypes to fruit rot caused by Ceratocystis radicicola in Iran

Mohammad R. Mirzaee^I; Mojtaba Mohammadi^II; Ali Azari Nasrabad^I

^IAgricultural and Natural Resources Research Center of Southern Khorasan, Birjand, P.O. Box 413, Iran

^IIDepartment of Plant Pathology and Microbiology, 1463 Boyce Hall, University of California, Riverside, CA 92507, USA

ABSTRACT

Several citrus genotypes were evaluated for their relative susceptibility to the new lemon fruit rot pathogen, Ceratocystis radicicola. Wounded detached fruits were inoculated ten days before normal harvest by placing on the wound site a droplet of distilled water followed by a mycelial plug of one-week-old culture. Inoculated fruits were ranked for their relative susceptibility to the pathogen by determining disease severity based on mean lesion size. Using Duncan's multiple range test, citrus varieties were classified into three groups, as follows: most susceptible: Mandarin (cv. Clementine); moderately susceptible: Mandarin (cvs. Dancy, Ponkan, sweet lime and common sour orange) and least susceptible: Mandarin (cvs. Kinnow, Lee, Fortune and Osceola), grapefruit (cvs. Marsh and Red Blush), orange (cvs. Parson Brown, Marss Early, Salustiana, Washington Navel and Hamlin) and lemon (cv. Lisbon). Alternatively, fruit firmness was measured using a hand-held penetrometer at the time of inoculation. Disease severity was negatively correlated (R = -0.36, P < 0.01) with fruit firmness. Although this study aimed to determine the range of potential hosts for C. radicicola, to date the only natural host in the world is considered to be lemon.

Keywords:Ceratocystis radicicola, Citrus susceptibility, resistance, fruit firmness.

Ceratocystis spp. constitute important pathogens of trees as well as seeming to be saprophytic species. Among the pathogenic species, C. fimbriata Ellis & Halst. has been reported as the causal agent of serious dieback in other citruses as well as lemon trees in Colombia and Argentina (Mourichon, 1994; Montoya & Wingfield, 2006; van Wyk et al., 2007). Fruit rot disease of lemon caused by Ceratocystis radicicola (Bliss) C. Moreau (anamorph Thielaviopsis punctulata (Hennebert) A.E. Paulin, T.C. Harr. & McNew) and Bacillus sp. was reported for the first time from Iran (Mirzaee et al., 2001a, b). C. radicicola has previously been shown to be the causal agent of sudden death of date palm (Phoenix dactylifera) in the USA and South Africa (Bliss, 1941; Linde & Smit, 1999). C. radicicola was also isolated from Pinus sp. in the United Kingdom and from the rhizosphere of date palm in Iran (Alavi & Sonbolkar, 2000; Jones & Baker, 2007). Further, several citrus species including Mexican lime, sweet lime, orange, sour orange, grapefruit, rough lemon and mandarin were evaluated for their susceptibility to C. radicicola fruit rot. The results showed that the other citrus species were susceptible to the fungal infection whereas immature lemon fruits and seedlings were resistant (Mirzaee & Mohammadi, 2005). Further, the intact citrus fruits were not susceptible to infection by fungal spores of C. radicicola (Mirzaee & Mohammadi, 2005). Fruit firmness has been correlated with fruit rot incidence as well as fruit susceptibility to pathogens such as Rhizopus fruit rot, Monilinia laxa and Botrytis cinerea (Daubeny et al., 1980; Bassi et al., 1998; Kempler et al., 2005; Kvikliene et al., 2006; Mirzaee et al., 2007). Fruit firmness was correlated with postharvest time and fruit maturity in orange varieties (Olmo et al., 2000). The objective of this study was to determine the relative susceptibility of various citrus genotypes to C. radicicola, the causative agent of fruit rot of lemon in Iran, under laboratory conditions, in order to evaluate the potential risk of the disease to other cultivars.

Fruits were harvested ten days prior to their normal harvest date from a citrus collection in Jiroft region (latitude/longitude: 28/67N, 57/73E), Southeastern Iran, thoroughly washed with non-chlorinated tap water and surface-sterilized with 75% (v/v) ethanol. Inoculation sites were cleaned with sterile cotton and allowed to dry in a chamber. To obtain the optimal conditions for fruit inoculation, the following methods were tested: A) the inoculum was prepared by suspending phialoconidia of one-week-old culture of C. radicicola in sterile distilled water to a concentration of 5×10⁵ spores/mL. Filter paper discs (5 mm diameter) were dipped in the inoculum and placed over the fruit rind; B) inoculum was prepared as described above, 100 µL suspension of phialoconidia was injected into the fruit rind and C) inoculating the fruits first by making a one-mm-deep wound through the fruit rind with a sterile 5 mm-diameter cork borer, followed by placing 10 µL distilled water in the wound site, and then filling with a 5 mm-diameter mycelial plug of a one-week-old culture grown on potato dextrose agar (PDA, Merck). Inoculated wounds were wrapped in parafilm to maintain moisture. Inoculated fruits were placed in a plastic box with a closed lid, and incubated at 25ºC for 3 days. For each cultivar, a total of 16 fruits were used, which included three independent replications (blocks) each consisting of four samples and four uninoculated controls on detached fruits. The experiment was performed as a completely randomized block design with 17 cultivars as treatments. The fungus was re-isolated periodically to confirm identity and its pathogenicity. Further, four additional fruits of each cultivar were selected for determination of fruit firmness at the time of inoculation (Biggs & Miller, 2004, 2005). Fruit firmness was measured using a hand-held penetrometer (Eijkelkamp, Netherlands) fitted with an 11-mm tip. Detached fruits were rated for disease severity by measuring length and width of each lesion three days after inoculation (Biggs & Miller, 2004). The results were analyzed using the analysis of variance (ANOVA). Mean lesion diameter data were subjected to a general linear model analysis and means were compared by Duncan's Multiple Range Test (MSTAT-C) at 1% probability level. The Spearman correlation analysis was used to determine the relationship between the disease severity and fruit firmness.

Two days after fruit inoculation, decay symptoms were observed on inoculated fruits. Control fruits showed no symptoms and no fungus was isolated from these tissues either. The causal agent was re-isolated from the inoculated fruits. Among various fruit inoculation tests, method C, which involved making a wound through the fruit rind and placing mycelial agar, was the most promising. Method A, which involved placing filter paper discs over the fruit rind, resulted in no disease symptoms. Likewise, method B, which involved the injection of phialoconidia suspension into the rind of fruits, was found to be unreliable due to the fact that the spore suspension was not distributed evenly into the flesh of a number of cultivars.

As shown in Table 1 and Figure 1, fruit firmness was negatively correlated with lesion development by C. radicicola on fruit rind (R = -0.36, P < 0.01). Fruit rot ranged from 6 cm in diameter for mandarin (Citrus reticulata) cv. Clementine to 0.74 cm for mandarin cv. Fortune. The former is considered the most susceptible cultivar and the latter the least. Based on Duncan's multiple range test, citrus cultivars were classified into three relative susceptibility groups: most susceptible: mandarin (cv. Clementine), moderately susceptible: mandarin (cvs. Dancy and Ponkan), sweet lime and common sour orange, and least susceptible: mandarin (cvs. Kinnow, Lee, Fortune and Osceola), grapefruit (cvs. Marsh and Red Blush), orange (cvs. Parson Brown, Marss Early, Salustiana, Washington Navel and Hamlin) and lemon (cv. Lisbon). There was no species of citrus that was more or less susceptible than the species used in this study.

Citrus fruits tested in this study represent a unique collection of cultivars from the same location in order to eliminate any variability due to environmental factors as well as soil texture on fruit quality. The results showed that the potential host range of C. radicicola is broader than that observed under natural infection conditions. The disease has been observed and reported on lemon, a natural host for C. radicicola, whereas under laboratory conditions, all citrus genotypes were found to be susceptible. Many genotypes investigated in this study were more susceptible to fruit rot than lemon. On the other hand, in spite of the existence of very susceptible genotypes, lemon fruit damage only occurs during the harvest as well as when branch thorns come into direct contact with fruits in southern Iran. This is the first study of citrus fruit rot on susceptible varieties in Iran. Therefore, a thorough investigation of its natural prevalence on other citrus cultivars, as well as fruit rot etiology and life cycle of the causative pathogen, are needed.

Fruit firmness has been demonstrated to be an important contributing factor for resistance (Daubeny et al., 1980; Bassi et al., 1998; Biggs & Miller 2004; Kvikliene et al., 2006; Blazec et al., 2007). On the other hand, fruit firmness was correlated with postharvest time and fruit maturity in orange varieties (Olmo et al., 2000). This characteristic suggests that fruit maturity is related to mechanical properties and fruit quality (Ladaniya 2008). Therefore, it is recommended that fruit firmness and its correlation to disease severity be evaluated. Negative correlations were also observed between fruit firmness in apple cultivars and Botryosphaeria spp., ideotypes of apple and storage diseases and red raspberry and Rhizopus fruit rot (Daubeny et al., 1980; Biggs & Miller, 2004; Blazec et al., 2007).

In this study, fruit firmness had a negative correlation with susceptibility of citrus genotypes to C. radicicola. We observed unexpected results in the case of fruit firmness correlating with susceptibility of mandarin cvs. Osceola, Lee and common sour orange (Figure 1). These discrepancies show that the interaction between the fungus and the citrus host is much more complex than was thought and, in addition to fruit firmness, other factors may play a role in disease development and host resistance. Expression of defense-related genes, pathogenesis-related proteins, phytoalexins, flavonoids and other secondary metabolites induced citrus fruit protection against fungal infections (Arcas et al., 2000; Ortuno et al., 2008). For instance, Ortuno et al. (2008) have suggested that ethylene may be considered as a possible marker for susceptibility of citrus fruits to Alternaria alternata pv. citri. Fortune, which is the most susceptible variety, produced more ethylene during growth than the less susceptible C. lemon and C. paradisi. Thus, further investigation is needed to unravel the mechanism(s) underlying the interaction between citrus fruits and C. radicicola.

C. radicicola is known to be a soil-borne pathogen (Wingfield et al., 1993). Poor sanitary conditions, fruit damage during harvest and uncut branches touching the soil surface in citrus groves may contribute to fungal infection and spread. Further, citrus and date palm trees, which are natural hosts of the pathogen, are planted together in Jiroft region (Mirzaee & Mohammadi, 2005). Under laboratory conditions, disease progress caused by C. radicicola on Mandarin fruit (cv. Dancy) was significantly greater than that caused by Alternaria sp. (Mirzaee & Mohammadi, 2005). Therefore, C. radicicola should be considered a serious threat to the citrus industry in the region and neighboring countries where citrus and date palm trees are planted together (Mirzaee & Mohammadi, 2005). Furthermore, C. radicicola may become a threatening pathogen in the US and South Africa, where it was originally reported.

There are several sanitary measures for managing citrus fruit rot disease. These include pruning citrus tree branches touching the soil surface, avoiding fruit damage, separating fruits that had contacted the soil from the rest and preventing the fruits from touching the soil. These measures are also advised for the control of Phytophthora fruit rot, Botrytis fruit rot and citrus sour rot (Whiteside et al., 1988). In order to alleviate disease severity due to fungal fruit rot, and to increase fruit firmness, it is highly recommended that fruit damage be prevented during harvest. Loss of citrus fruit firmness has been correlated with the degradation of the cell wall (Olmo et al., 2000). It is well known that calcium has a major effect on enhancing fruit firmness, cell wall structure and reducing the incidence of physiological disorders and decay (Salcedo et al., 2000; Malakouti & Tabatabaee, 2001; Kader & Rolle, 2004). High nitrogen content and excess water supply to plants, on the other hand, are often associated with reducing fruit firmness and increasing susceptibility to mechanical damage and decay (Kader & Rolle, 2004). Therefore, it would also be possible to use fertilizers to boost plant nutrition, aiming at enhancing fruit resistance to mechanical injuries.

Received 27 March 2009

Accepted 23 November 2009

Author for correspondence: Mohammad R. Mirzaee, e-mail: [e-mail]

Section Editor: Francisco F. Laranjeira

• Alavi A, Sonbolkar A (2000) Dry rot of date bunch. Proceedings of the 14^th Iranian Plant Protection Congress, Isfahan University of Technology, Isfahan, Iran.
• Arcas MC, Botia JM, Ortuno AM, Del Rio JA (2000) UV irradiation alerts the levels of flavonoids involved in the defence mechanism of Citrus aurantium fruits against Penicillium digitatum European Journal of Plant Pathology 106:617-622.
• Bassi D, Rizzo M, Cantoni L (1998) Assaying brown rot (Monilinia laxa) susceptibility in peach cultivars and progeny. IV^th International Peach Symposium, Bordeaux, France.
• Biggs AR, Miller SS (2004) Relative susceptibility of selected apple cultivars to fruit rot caused by Botryosphaeria obtusa HortScience 39:303-306.
• Biggs AR, Miller SS (2005) Comparative relative susceptibility NE-183 apple cultivars to fruit rot pathogens in west Virginia. Journal of American Pomological Society 59:72-77.
• Blazek J, Opatova H, Golias J, Homutova I (2007) Ideotype of apples with resistance to storage diseases. Horticultural Science (Prague) 34:107-113.
• Bliss DE (1941) Relation of Ceratostomella radicicola to rhizosis of the date palm. Phytopathology 31:1123-1129.
• Daubeny HA, Pepin HS, Barritt BH (1980) Post-harvest Rhizopus fruit rot resistance in red raspberry. HortScience 15:35-37.
• Jones DR, Baker RHA (2007) Introductions of non-native plant pathogens into Great Britain, 1970-2004. Plant Pathology 56:891-910.
• Kader AA, Rolle RS (2004) The role of post-harvest management in assuring the quality and safety of horticultural produce. FAO Agricultural Services Bulletin 152.
• Kempler C, Daubeny HA, Harding B, Finn CE (2005) Esquimalt red raspberry. HortScience 40:2192-2194.
• Kvikliene N, Kviklys D, Viskelis P (2006) Changes in fruit quality during ripening and storage in the apple cultivar Auksis. Journal of Fruit and Ornamental Plant Research 14 (Suppl. 2):195-292.
• Ladaniya MS (2008) Citrus fruit: Biology, technology and evaluation. San Diego CA. Elsevier Academic Press.
• Linde C, Smit WA (1999) First report of rhizosis caused by Ceratocystis radicicola on date palms in South Africa. Plant Disease 83:880.
• Malakouti MJ, Tabatabaei SJ (2001) Innovative approach to balanced nutrition of fruit trees. Tehran, Iran. Sana Press.
• Mirzaee MR, Ershad D, Rahimian H, Mohammadi M (2001a) A new report of Ceratocystis radicicola to cause lemon fruit rot in Iran. Proceedings of the Asian International Mycological Congress, Karaj, Iran.
• Mirzaee MR, Ghasemi A, Rahimian H, Mohammadi M (2001b) A new report of Bacillus sp. lemon fruit rot in Iran. Iranian Journal of Plant Pathology 37:47.
• Mirzaee MR, Mohammadi M (2005) A new report of fruit rot of lemon caused by Ceratocystis radicicola from Iran. Journal of Phytopathology 153:11-14.
• Mirzaee MR, Safarnejad MR, Mohammadi M (2007) A new report of pre-harvest ear rot of corn caused by Geotrichum candidum from Iran. Communications in Applied Biological Sciences 72:925-933.
• Montoya MM, Wingfield MJ (2006) A Review of Ceratocystis sensu stricto with special reference to the species complexes C. coerulescens and C. fimbriata Revista Facultad Nacional de Agronomía, Medellín 59:3045-3075.
• Mourichon X (1994) Serious citrus dieback in Colombia caused by Ceratocystis fimbriata Fruits (Paris) 49:415-416.
• Olmo M, Nadas A, Garcia GM (2000) Nondestructive methods to evaluate maturity level of oranges. Journal of Food Science 65:365-369.
• Ortuno A, Nemsa I, Alvarez N, Lacasa A, Porras I, Garcia Lidon A, Del Rio JA (2008) Correlation of ethylene synthesis in Citrus fruits and their susceptibility to Alternaria alternata pv. citri. Physiological and Molecular Plant Pathology 72:162-166.
• Salcedo F, Matta F, Killebrew F, Garner JO (2000) Influence of nitrogen and calcium fertilizer on fire blight susceptibility of Royal Gala apple trees. Mississippi Agricultural and Forestry Experiment Station, USA. Bulletin 1093.
• van Wyk M, Pegg G, Lawson S, Wingfield MJ (2007) Ceratocystis atrox sp. nov. associated with Phoracantha acanthocera infestations on Eucalyptus grandis in Australia. Australasian Plant Pathology 36:407-414.
• Whiteside JO, Gransey SU, Timmer LW (1988) Compendium of citrus diseases. Saint Paul MN. APS Press.
• Wingfield MJ, Seifert KA, Webber JF (Eds.) (1993) Ceratocystis and Ophiostoma Saint Paul MN. APS Press.

Publication Dates

• Publication in this collection
  04 Jan 2010
• Date of issue
  Oct 2009

History

• Received
  27 Mar 2009
• Accepted
  23 Nov 2009