Effects of temperature and leaf wetness duration on pathogens causing preharvest fruit rots on tomato

Preharvest fruit rots are serious tomato diseases. This study showed that Botrytis cinerea was a significant problem, and that Alternaria (A. alternata, A. solani) was the main pathogen identified from tomatoes showing the symptoms of preharvest fruit rots. Fusarium oxysporum and Rhizoctonia solani were also found causing important preharvest fruit rots in tomatoes. This is the first time F. oxysporum and R. solani have been identified in Greece as pathogens causing preharvest fruit rots on tomatoes. The optimal temperatures for A. alternata, A. solani, B. cinerea, and F. oxysporum mycelial growth and conidial germination were generally found to be between 20 and 30 °C, while the wetness duration of 24 hours seemed to be optimal for conidial germination and 84 hours for R. solani sclerotial germination. Based on the above findings, logistic regression models that adequately described the impact of pre-inoculation temperature and wetness on infection incidence in tomato fruit can be created. As the minimum, maximum, optimal temperatures and wetness duration are generally similar for the main pathogens causing preharvest fruit rots in tomato, it is possibly to develop a predicting predictive model to forecast the preharvest infection of tomato fruit. So enabling advice to be given to growers to when control methods should be applied.


Introduction
Tomato (Lycopersicon esculentum Mill.) is one of the most economically significant vegetable crops in Greece where it is grown both indoors and outdoors.Greece produced 835940 tonnes of tomatoes in 2018, from a total harvested area of 16020 hectares (FAOSTAT 2022).Fruit rots are of the most economically important diseases in tomato crops, which are caused by a number of plant pathogens.These diseases can destroy tomatoes in field or/and during storage.Thus, the recognition of symptoms and the knowledge of the distribution and spread of the pathogens causing fruit rots in tomatoes and of the methods for early detection of the pathogens are the major prerequisites for successful management of these diseases.Previous works showed that 10-30% of the total yield of perishable crops can be destroyed by pre and post harvest diseases, especially under favorable environmental conditions (Agrios 2005;Kader 2002).The pathogenic fungi may be responsible for unsafe food by producing mycotoxins, in addition to rots (Moss 2002).
In Nigeria, Fajola (1979) identified two forms of tomato fruit rots: the soft rot was found responsible for 85% of the total loss, and the dry rot for approximately 15%.Additionally, he found that pathogens causing soft rot in tomato fruits include Erwinia carotovora, Rhizopus oryzae, R. stolonifer, Fusarium equiseti, F. nivale, and F. oxysporum, whereas those that cause dry rot in tomato fruits include Aspergillus aculeatus, A. flavus, Cladosporium tenuissimum, Corynespora cassiicola.Oladiran and Iwu (1993) identified seven fungi, including F. equbeti, F. chlamydosporum, A. solani, G. candidum, A. recifei, A. flavus, and A. niger, that are associated to tomato fruit rot.
Meteorological factors play an important role in the development of fruit rots.The main factors influencing the development of these fungi are temperature and moisture.As a result, understanding the favorable temperatures and moisture for pathogen development is critical in determining the methods and timing of its control (Miller 1953).The main aims of this study were to identify pathogens causing preharvest fruit rots on tomato in Northern Greece.In addition, the effect of temperatures and wetness on the mycelial growth and conidia germination of the identified pathogens from rotted tomatoes was investigated.Mathematical models were developed that adequately described effects of wetness duration and temperature on infection incidence in tomato fruit.

Identification of pathogen
In June 2018 and again in June 2019, two hundred ripe tomatoes showing symptoms of preharvest fruit rots were collected from outdoor commercial fields, half from the area of Nea Magnisia Thessaloniki and the other half from the area of Naoussa Imathia.Certain cultural practices including single plant staking, irrigation, fertilizations and pest control were applied at both sites.A meteorological station was established in each area to record air temperature and relative humidity during the period of May-June 2018 and again 2019 (Figs. 1 and 2).The samples included fruits with total surface area rough but firm, fruits with total surface area rough and slightly depressed, fruits with total surface area rough and depressed, fruits with total surface area rough and easily depressed and fruits with total surface area very rough and fruit depressed with slightest pressure.The fruit were transferred in the laboratory of Mycology and Food Technology of International Hellenic University.Fruits were disinfested for 2 minutes in 10% solution of domestic bleach, rinsed with sterile distilled water multiple times, and dried using sterile filter sheets.To isolate the pathogens, lesions were aseptically cut, plated on sterile potato dextrose agar (PDA) in 9 cm Petri plates and incubated at 23 ºC for 3 to 7 days.Isolates were first identified to genus based on comparison of morphological characteristics of the mycelium, sclerotia and spores and sporeproducing structures with descriptions available in the literature (Barnett and Hunter 1998;Pitt and Hocking 2009).Pure cultures of isolates from all fungal genus, except Alternaria spp., were transferred to fresh PDA and incubated at 23 °C with a 12-h photoperiod for up to 4 weeks to induce sporulation.Isolates tentatively identified as Alternaria spp.were grown on V8 juice agar.The species of the isolated fungi was identified by using the internal transcribed spacer region (ITS) of ribosomal DNA with the universal primers ITS1 and ITS4.Fungal DNA was extracted from freshly collected mycelium of 7-day-old cultures using the DNA extraction Kit (QIAGEN DNA Mini Kit, HB-1166, Hilden, Germany).The internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers ITS1 and ITS4.The forward primer ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and reverse primer ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3').The ITS sequence was compared with the NCBI GenBank database sequences using the BLAST search tool (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi) (Table 1).The analyses supported the results obtained in the morphological study.
Two different isolates of each of the above pathogens were used to complete Koch's postulates.Mature tomatoes (ten for each isolate) were surface sterilized in 0.1% chlorine in the lab before each tomato was artificially inoculated with a fungal isolate by placing a mycelial disk into the fruit's wound site.As a control, tomatoes were inoculated with agar disks without mycelium.

Effect of temperatures on the mycelial growth and conidia / sclerotia germination
Mycelia Growth: A mycelial agar disk (6 mm in diameter) of each Alternaria (A. alternata, A. solani), B. cinerea, R. solani and F. oxysporum, identified above, were collected from the edge of an active colony, put in the middle of dishes (9 cm in diameter) containing PDA, and incubated in a growth chamber at 0 (refrigerator), 5,10,15,20,25,30,35, and 40 °C.The diameter of the developed colony was measured in order to gather data 5 days later.Two randomly selected isolates from each fungal pathogen were used.There were four replicated Petri dishes for each isolate.This experiment was repeated once.
Sclerotial Germination: To produce sclerotia of R. solani, the method described by Ritchie et al. (2009) was used.Two randomly selected isolates came from the above rotted tomatoes were used.Sclerotia were collected aseptically by scraping off the surface of cultures that had been growing for 21 days at 25 °C on solid malt yeast extract media.Harvested sclerotia were put in fine mesh nylon bags, and agar fragments were removed by washing the bags with sterile distilled water.Washed sclerotia were transferred onto Whatman no.1 filter papers then placed in sterile plastic Petri dishes and left to dry in a laminar flow cabinet.The effect of temperature on sclerotial germination was investigated by placing ten sclerotia aseptically onto 9 cm Petri dishes containing PDA and incubated in a growth chamber at 0 (refrigerator), 5 10, 15, 20, 25, 30, 35, 40 °C for 72 h.Germination was determined by assessing individual sclerotia for outgrowing hyphae under a microscope at 45X magnification.A sclerotium was considered to have germinated when outgrowing hyphae were equal to or greater than its diameter and the percentage of sclerotia germinated per plate recorded.There were 4 replicated Petri dishes for each treatment.This experiment was repeated once.Conidia Germination: Conidia were obtained from lawn cultures of each Alternaria (A. alternata, A. solani), B. cinerea, and F. oxysporum on PDA by drenching the culture with sterile distilled water that contained 0.01% (v/v) Tween 20 wetting agent (BDH Ltd., Poole, UK) and dislodging spores from the hyphae with the aid of a sterile glass spreader.By passing the spore-containing solution through two layers of moistened, sterile cheesecloth, any remaining hyphal fragments were removed.The number of spores was adjusted to 10 5 conidia / ml counted using a hemocytometer, and stock spore solution was kept at 4 °C until use.One ml of the suspension was added to PDA-filled Petri dishes before being spread out and left in a growth chamber with constant moisture for 24 hours at 0 °C (the refrigerator), 5 °C, 10 °C, 15, 20 °C, 35 °C, and 40 °C.Counting 100 spores in each Petri dish under a microscope at 400x was used to calculate the percentage of conidia germination.When the length of the germ tube exceeded the spore's diameter, the conidium was considered to have germinated (Dantigny et al. 2006;Dhingra and Sinclair 1985).There were 4 replicated Petri dishes for each treatment.This experiment was repeated once.

Effect of wetness on the conidial / sclerotial germination
Conidia were harvested from lawn cultures of each Alternaria (A. alternata, A. solani), B. cinerea and F. oxysporum on PDA as above.The number of spores was adjusted to 10 5 conidia / ml counted using a hemocytometer.One ml of the suspension was spread in Petri dishes containing PDA and placed in a growth chamber under continuous wetness at 25 °C, for 3, 6, 9, 12, 24, 36 and 48 h.A wet sterilized paper towel was stuck at the down side of the lid of the Petri dish to keep high moisture.The percentage of the germinated spores was recorded as described above (Dantigny et al. 2006;Dhingra and Sinclair 1985).There were 4 replicated Petri dishes for each treatment.This experiment was repeated once.
Similarly, sclerotia of R. solani were harvested as described above.The effect of wetness on sclerotial germination was investigated by placing ten sclerotia aseptically onto 9 cm Petri dishes containing PDA and incubated in a growth chamber at 25 °C, under 90-95% RH, for 36, 48, 60, 72, 84, 96 h.A wet sterilized paper towel was stuck onto down side of the lid of the Petri dish to keep high moisture.Germination of sclerotia was determined as described above.There were 4 replicated Petri dishes for each treatment.This experiment was repeated once.

Statistical analysis and modeling of pathogen development
The effect of temperatures and wetness on mycelial growth and conidial / sclerotial germination was determined by analysis of variance (ANOVA) for α=0.05.Normality of data was tested and, when needed, data were transformed appropriately to avoid heterogeneity of variances (Sokal and Rohlf 1995).Non-linear regression models were used to fit the data to illustrate and simulate the relationship between the abiotic factors (i.e.temperature and wetness duration) on the developmental events (mycelial growth or conidial / germination) for each pathogen.
Temperature effect was modeled according to the following three-parameter Gaussian type model: While, leaf wetness duration was modeled according to a Gompertz type model given as follows: where, x is the factor under consideration and α, b and c constant parameters.Parameter estimates were estimated based on the Levenberg-Marquardt algorithm (LMA) which provides a numerical-iterative solution of curve fitting over a space of parameters of the function by sting first some initial values serving as starting point for the iteration (Damos and Savopoulou-Soultani 2012).The final parameters of the model curve were fixed so that the sum of the squares of the deviations for each value pairs, x i and y i is minimum: To test the goodness of fit of the nonlinear models the following model performance statistics were estimated: The coefficient of determination and the related adjusted R 2 .The adjusted R 2 (Adj.Rsq.)is: . where: n is the number of points in the data set, k is the number of independent variables in the model, excluding the constant and R 2 = Explained Variation/Total Variation is the coefficient of determination.
The F-test of overall significance, to determine how well the non-linear regression models obtained fits the given data points.The statistic is: where df ssr are the degrees of freedom for the regression model; df sse are the degrees of freedom for the error term.This value is equal to the total number of data records n minus the number of coefficients p: df sse = n -p -1.The resulting F statistic was compared to an F-table to extract the p-value to determine how much uncertainty there is in the values of the parameters and for α=0.05 significance level.The alternative hypothesis is that, contrary to the null hypothesis, the data were produced in accordance with the model being fitted with certain parameter values that were unknown.
The Shapiro-Wilk statistic (WS) additional applied to determine how evenly distributed the observational data are around the regression curve (Royston 1992).The formula of the WS is: where x i are the ordered random sample values and α i are constants generated from the covariance's, variances and the sample means.
The parameter values of the non-linear regression equations and the related model performance statistics were estimated using r-computing language (Ritz et al. 2015;Core team 2015). 2

Identification of pathogen
To fulfill Koch's postulates, the above fungi were re-isolated from artificially inoculated tomatoes that displayed symptoms similar to those observed on naturally infected tomatoes in the commercial field.
The results of this study showed that A. alternata and A. solani were the main pathogens found in ripe tomato fruits showing symptoms of rotting, while the fungus B. cinerea was also identified in a relatively high percentage both 2018 and 2019 (Fig. 3).This study also showed that a significant proportion of tomatoes with preharvest fruit rot symptoms were caused by the fungi R. solani and F. oxysporum.No pathogen was identified from a percentage of 6% and 4% of the total rotted tomatoes in 2018 and 2019 respectively, as the calcium deficiency was possibly the cause.
The climate conditions in Nea Magnisia and Naoussa Imathia in the period of June 2018 and 2019 were with temperatures fluctuated between 15-30 °C, and continuously periods with high value of leaf wetness, which accounts for the preharvest infections of tomato fruits from the pathogens A. alternata, A. solani, B. cinerea, R. solani and F. oxysporum.

Effect of temperatures and wetness on mycelial growth and conidial / sclerotial germination
There was no significant difference between repeated trials, so the data from the two trials were combined.
Rhizoctonia solani: Temperature significantly influenced mycelial growth (P<0.000,SE= 0.133).The optimum temperature for mycelial growth was 25-30 °C, gradually reduced at 15 °C and 35 °C, whereas mycelial growth was inhibited at 10 °C and 40 °C.The estimates of  2 and Fig. 5.It was also found that as wetness duration increased up to 84 h, there was an increase in the percentage of sclerotial germination, but further wetness duration up to 96 h did not further increase the percentage of sclerotial germination (Table 2; Fig. 6).

Botrytis cinerea:
The results showed that temperature significantly influenced both the mycelial growth (P<0.001,SE= 0.166) and the conidia germination (P<0.000,SE= 0.266).Maximum mycelial growth and conidia germination of B. cinerea on PDA was between 20-25 °C, gradually reduced at 5 °C and 35 °C and inhibited at 0 °C and 40 °C (data are not presented).The estimates of the parameters are presented in Figs.7 and 8.The results of this study also showed that as wetness duration increased up to 24 h, there was an increase in the percentage of conidia germination of B. cinerea, but further wetness duration up to 48 h did not further increase the percentage of conidia germination (Fig. 9).Conidia of B. cinerea did not germinate at wetness duration shorter than 6 h.Fusarium oxysporum: Temperature significantly influenced both the mycelial growth (P<0.004,SE= 0.230) and the conidium germination (P<0.014,SE= 0.202).The results showed that the optimal mycelial growth was observed between 20-30 °C, gradually reduced at 10 °C than 35 °C and inhibited at 5 °C and 40 °C.The highest percentage of conidia germination was found between 25-30 °C, gradually reduced at 5 °C and 35 °C and inhibited at 0 °C and 40 °C.The estimates of the parameters are presented in Table 2 gradually increased as the wetness period increased from 6 to 12 h, but not further increase was observed up to 48h (Table 2, Fig. 12).No conidia germination of F. oxysporum was observed at wetness duration up to 3 h.Alternatia alternata: It was found that temperature significantly influenced both the mycelial growth (P<0.006,SE= 0.215) and the conidium germination (P<0.002,SE= 0.237).Highest mycelial growth was observed between 25-30 °C, gradually reduced at 5 °C and 35 °C and inhibited at 0 °C and 40 °C.The highest percentage of conidia germination was found at 25 °C, gradually reduced at 10 °C than 35 °C and inhibited at 5 °C and 40 °C.The estimates of the parameters are presented in of Alternaria alternata gradually increased as the wetness period increased from 6 to 24 h, but no further increasing was observed up to 48h (Table 2; Fig. 15).No conidia germination of A. solani was observed at wetness duration up to 6 h.
Alternatia solani: Temperature significantly influenced both the mycelial growth (P<0.001,SE= 0.202) and the conidium germination (P<0.004,SE= 0.251).Maximum mycelial growth and conidia germination was observed at 25 °C, gradually reduced at 10 °C and 35 °C and inhibited at 0 °C and 40 °C.The estimates of the parameters are presented in Table 2, Figs. 16 and 17.This study also showed that increasing of the wetness duration from 12 to 24 h, increased the percentage of conidia germination of A. solani, and while further increasing of the wetness duration did not cause a significant increase.(Table 2, Fig. 18).No conidia germination of A. solani was observed at wetness duration up to 6 h.

Discussion
There has been a lot of research done on the pathogens that cause tomato fruit rots after harvest, but relatively little is known about the causes of fruit rots before harvest.
It is well-know that fruit ripening is a natural process that occurs during the final stages of fruit development and is associated with changes in color, texture, aroma, and flavor.However, as fruits ripen, they also become more susceptible to various pathogens (Prusky 1996).The increased susceptibility of ripe fruit to opportunistic pathogens is likely to facilitate the dispersal of mature seed (Gillaspy et al. 1993;Prusky 1996).According to the study's findings, A. alternata and A. solani were the two most important pathogens found in rotten, ripe tomato fruits, while B. cinerea was also found in a quite high number of cases.Similarly, on tomatoes produced in a greenhouse in Suwon, Korea, black mold caused by the fungus A. alternata was the main disease (Kim et al. 2020).According to Butler (1959), black mold of processing tomatoes, which is caused by the fungus Alternaria, reduces production when heavily infected fruit rots and falls off the vine or truck loads with an 8% or higher incidence of mold are rejected at industry grading stations.Williamson et al. (2007) identified B. cinerea as one of the greenhouse tomato pathogen that causes the greatest losses in fruit throughout the preand post-harvest periods, and  . alternata, A. solani, B. cinerea, R. solani and F. oxysporum showed that their optimal growth temperatures fluctuated between 20-30 °C.Similarly, Swart and Holz (1991) found that the germ-tube growth of A. alternata was rapid at 20-30 °C, moderate at 10°C, but virtually non-existent at 0 and 5°C.They also reported that the highest mycelium growth of A. alternata was observed at 25 °C, while the radial growth at 10 °C was approximately 30% of the maximum, while Pennypacker, (1988) showed that conidia   Swanson and Van Gundy (1985) showed that the pathogen F. oxysporum f. sp.tracheiphilum grows well at temperatures ranging between 24 and 27 °C.This study also showed for the first time the importance of the duration of wetness period on spore germination of the pathogens A. alternata, A. solani, B. cinerea, F. oxysporum and sclerotium germination of the pathogen R. solani isolated from tomato fruit with symptoms of preharvest fruit rots.Specifically, more than 6 h duration of wetness period were required for the spore germination of A. alternata A. solani, B. cinerea and F. oxysporum.Different studies showed that the levels of infection Minneola Tangelo leaves by Alternaria sp. were low at 4 and 8 h of leaf wetness and continued to increase with longer wetting periods up to 36 h (Canihos et al. 1999), while Timmer et al. (1998) reported that conidial production of A. alternata was greatest on mature leaves moistened and maintained at near 100% relative humidity (RH) for 24 h.Vloutoglou and Kalogerakis (2001) studied the effect of leaf wetness in incubation period showing that 4 hours of leaf wetness after inoculation were sufficient to initiate of early blight (Alternaria solani) on hybrid Skala RZ plants but not on cv.Rio Rojo plants, which required at least 6 hours of leaf wetness.They also found that there was an increase in the percentage of leaf area showing symptoms and the percentage of defoliation as wetness duration increased up to 24 h, but no significant increase in either parameter after that.In other studies, Sirjusingh and Sutton (1996) studied the effect of wetness duration on sporulation of B. cinerea showing that sporulation incidence of B. cinerea in whole flowers inoculated with conidia increased sharply when wetness duration at 15, 21, 25, and 30 °C was increased from 8 to 24 h, 4 to 12 h, 4 to 12 h, and 4 to 6 h, respectively.The results of this study also showed that as the duration of wetness period increased from 48 to 84 h, there was an increase in the percentage of sclerotial germination of R. solani, but further increasing of the wetness duration up to 96 h did not further increase the percentage of sclerotium germination.While the effect of soil moisture on sclerotial germination of R. solani has been extensively investigated, there is not information on the role of wetness on the infection of aerial parts of plants from the above fungus.

Conclusions and recommendations
Generally, this study showed that the fungi of genus Alternaria (A. alternata, A. solani) were the main pathogens causing preharvest fruit rots in tomatoes, while the fungus B. cinerea was also important.The fungi F. oxysporum and R. solani were also responsible for a quite high percentage of the total rotted tomatoes in both years.This is the first report identified F. oxysporum and R. solani as pathogens causing preharvest fruit rots of tomato in Greece.
Although the in vitro studies reported here do not directly simulate the conditions of the natural environment, the results provide an insight to the likely behaviour and growth of the main pathogens causing preharvest fruit rot in tomatoes.Generally, the optimal temperatures for the mycelial growth and conidia germination of A. alternata, A. solani, B. cinerea, R. solani and F. oxysporum was observed at 20-30 °C, while the wetness duration 24 h seems to be the best for conidia germination and 84 h for sclerotial germination.Based on above results, mathematical models can be developed that adequate described the effects of pre-inoculation wetness and temperature on infection incidence in tomato fruit.As the minimum, maximum, optimal temperatures and wetness duration is generally similar for the main pathogens causing preharvest fruit rots in tomato, it is possible to develop a predicting model to forecast the infection of mature tomato fruit and to advise growers when controlling methods must be applied.
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Fig. 1
Fig. 1 Mean air temperatures in the areas of Nea Magnisia and Naoussa Imathia in the periods of May-June 2018 and 2019

Fig. 3
Fig.3Pathogens causing postharvest fruit rots in tomato.The frequencies are calculated as the percentage of each isolated pathogen from the 100 sampled tomato fruit in 2018 and again in 2019.One pathogen was isolated from each fruit.No pathogen was isolated from 6 and 4 fruits in 2018 and 2019 respectively

Fig. 9
Fig. 9 Effect of wetness duration on the percentage of the in vitro conidial germination of Botrytis cinerea

Fig. 11 Fig. 12
Fig. 11 Effect of temperature on the percentage of the in vitro conidial germination of Fusarium oxysporum

Fig. 13
Fig. 13 Rate of the in vitro mycelial growth of Alternaria alternata at different temperatures

Fig. 17
Fig. 17 Effect of temperature on the percentage of the in vitro conidial germination of Alternaria solani

Table 1
Identification of fungal isolates of ITS region of rRNA gene sequence (BLAST analysis)

Table 2
Non-linear model parameter estimates and related performance statistics in respect to different plant pathogens and developmental events as affected by different constant temperatures and leaf wetness duration , Figs. 10 and 11.According to the results of this study, the percentage of conidia germination of F. oxysporum

Table 2 ,
Figs. 13 and  14.It was found that the percentage of conidia germination Bakar et al. (2013)anked B. cinerea second among fungal pathogens of economic significance.This study is the first report of R. solani and F. oxysporum as pathogens causing preharvest fruit rots of tomato in Greece, despite the fact that the fungus F. oxysporum has been previously identified as the causal agent of Fusarium Wilt in tomato plants.According toBakar et al. (2013), F. oxysporum can cause tomato postharvest fruit rot.R. solani is a significant soil-borne fungus